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Select options from the Categories on the right to filter the list of Questions & Answers related to a specific topic, product, or technology. To clear a filter, click Start Over. Click a specific question to show its answer or click expand all to show all the answers on the page.
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In the EcoStudy software click the Results tab. The well table on the right side of the window contains the quantification results. Quantity denotes the calculated quantity of each individual sample, and Average Quantity denotes the average quantity of all the replicates.
You might need to resize or scroll the table to see all rows and columns. To resize the width, drag the vertical grey bar separating the table from the graph.
For some library prep, AMPure XP beads are user-supplied from Beckman-Coulter Genomics. See the appropriate TruSeq DNA or RNA library prep guide for more information.
Yes. Just like v2 kits, MiSeq v3 kits are compatible with all Illumina sample prep kits. Some sample prep protocols require slight changes to reach optimum clusters. A concentration of 4 nM is recommended to begin the denature and dilute step before loading libraries for sequencing. Check the Illumina Tech Support Bulletin Board for the latest information on updates to sample prep protocols.
All NextSeq 500 kits include reagents for paired-end sequencing and a paired-end flow cell. However, single-read runs are possible on a paired-end flow cell when the run is set up as a single-read run.
Nextera Rapid Capture Enrichment index 1 (i7) and index 2 (i5) sequences are identical to index sequences used in other Nextera based kits. They use the Read 1 (HP10), Read 2 (HP11) and Index 1 (HP12) sequencing primers.
Although index sequences are identical across Nextera kits, the index 2 primer reagents (i5, E501-E502) are not interchangeable across kits. Also, the Nextera Rapid Capture Enrichment kits use only two index 2 (i5, E501-E502) sequences compared to eight index 2 (i5) sequences used in other Nextera kits.
The specificity of any Real-Time PCR assay, whether TaqMan probe or SYBR Green I, is determined by the quality of the assay design. Non-specific amplification can occur for both SYBR Green I or TaqMan probe methods if the assay design is poor.
TaqMan assays will not generate a signal for any non-specific amplification whereas SYBR Green I assays might. However, non-specific amplification will affect the amplification efficiency and sensitivity of TaqMan assays in the same way as SYBR Green I assays, even though the amplification is not detected.
When designing primers for either system, it is important to avoid primer sets that generate any non-specific amplification products. With SYBR Green I assays, the ability to perform melt curve analysis is advantageous for primer design, as any non-specific amplification can be detected and identified in the melt curve. For TaqMan assays, detecting non-specific amplification usually requires another post-PCR analysis method such as agarose gel electrophoresis of the PCR products. Alternatively, the primers could be used in PCR with SYBR Green I and melt curve analysis performed after amplification to determine if any non-specific amplification occurs. This optimizes the primer design without the expense of a labeled probe.
No. These kits are for use on HiSeq and HiScanSQ only.
Sequencing runs of these library types are compatible with rapid run reagent kits on a HiSeq 2500 in rapid run mode. However, Illumina does not currently provide software for analyzing this type of data.
All validated Illumina library prep methods are supported to run on the HiSeq. However, make sure that you match samples with the desired output to maximize workflow efficiency.
TruSeq DNA PCR-Free and TruSeq Nano DNA LT library prep kit adapters are methylated. However, TruSeq HT library prep kit adapters are not methylated and are therefore not suitable for bisulfite sequencing applications.
The protocol includes one Zymo cleanup step (following "tagmentation") and one Ampure XP size selection step (following limited cycle PCR). The final library should have a median insert size of ~250-300 to support long paired end 2x150 read lengths on the MiSeq system.
The quality of DNA isolated from FFPE samples can be highly variable. Due to this variability, it is difficult to predict the quality of a library prepared from FFPE samples using the TruSight One protocol. Illumina does not support FFPE or degraded DNA as input. FFPE samples can be attempted using the TruSight One protocol, but failed libraries originating from this sample type are not eligible for replacement or troubleshooting by Illumina.
Yes, these libraries can be used on all HiSeq, MiSeq, HiScanSQ and GA instruments.
Yes, VariantStudio v2.1projects are compatible with VariantStudio v2.2. Samples, annotations, including any imported custom annotations, and filter favorites are all carried over to VariantStudio v2.2.
No. The MiSeq uses a flow cell that is specifically designed for the MiSeq.
The adapter plate is not compatible with TruSeq RNA v1 or v2 kits as the protocol and the reagents are different.
The same controls are used, but at different concentrations.
Eco does not use PMTs or photodiodes, it uses a CCD camera as the detector which is designed to compensate for dark current.
The 48-well plates used in the system are custom designed for the Eco system, so you cannot substitute other plates or 8-tube (0.2 ml) strips. The plates are in a six-by-eight-well format with the same pitch and well size as standard 384-well Real-Time PCR plates. This means that you can use multichannel pipettes compatible with 384-well plates with the Eco plates.
Eco plates and seals are only available through Illumina.
The TruSeq Nano DNA Library Prep Kit uses the same workflow as TruSeq DNA Sample Prep Kit. No changes have been made in analysis.
The TruSeq ChIP Sample Prep kit does not contain in-line controls due to the low input amount.
VariantStudio requires the installation of .NET Framework 4.0 (Full, not 4.0 Client Profile). If .NET 4.0 is not detected, the installer requests that you install the framework. Another prerequisite for installation is the use of a 64-bit version of Windows.
You need to increase your starting template concentration. However, the starting template volume (120 µl) is unchanged from previous kits.
Yes. The HiSeq X Reagent Kit v2 introduces a library denaturation step during reagent preparation before clustering on the cBot. The denaturation step coupled with enhanced exclusion amplification (ExAmp) reagents enables sequencing of TruSeq DNA PCR-Free libraries on the HiSeq X system, in addition to superior data quality and coverage.
Yes. There are two new reagents in MiSeq v3 kits: new Incorporation Mix (IMT) and new Scan Mix (USM), and the PR2 volume is increased to 500 ml to support longer runs.
Testing has shown that performance shows minimal variability when combining low numbers of indexes, or across particular combinations of indexes, so any number or combination may be used.
Testing has shown that performance shows minimal variability when combining low numbers of indexes, or across particular combinations of indexes, so any number or combination may be used.
No, the cBot plates are the same; however, if sequencing TruSeq HT libraries on single-read flow cells, the TruSeq Dual Index Sequencing Primer Box, Single Read (single use box) (catalog# FC-121-1003) is necessary in order to sequence both index reads.
Samples are checked for duplicate barcode numbers, and duplicates are not allowed. This does not prevent the same sample from being run on different barcodes. There is not a check performed at the genotype level to ensure unique samples. Additionally, different sources may have run the same commercially available sources of DNA and submitted the data. Therefore, we recommend performing genotype-level comparison of the data that you are downloading prior to analysis to ensure the most accurate frequency information for your study.
There are no in-line controls for the TruSeq Small RNA Library Prep Kit.
There are no in-line controls for the small RNA kit.
There are no fixed guidelines for variant filtering in VariantStudio. Appropriate filter settings depend on the intended use of the assay and must be determined for each project. See the VariantStudio User Guide for more information on the filtering options.
There are no restrictions; however we recommend that the Maximum Number of clusters is set to match the biology of your samples.
No. MiSeq Reporter and the Illumina Amplicon Viewer are used to view MiSeq data. For an overview of the software, see the MiSeq System software page.
No. HiSeq v2 and TruSeq rapid SBS reagents cannot be combined. Each set of reagents has been formulated specifically for use only with its respective flow cell and instrument run parameters. The cluster kits and flow cells are also paired with respective kit types and cannot be used interchangeably.
No. HiSeq v4 and TruSeq v3 high output SBS reagents cannot be combined. Each set of reagents has been formulated specifically for use only with its respective flow cell and instrument run parameters. The cluster kits and flow cells are also paired with respective kit types and cannot be used interchangeably.
Currently the ramp rate is not adjustable. Check back periodically and keep an eye on the monthly Illuminotes newsletter for updates.
Yes, this can be adjusted using Microsoft Excel. Information in any of the rows can be edited or deleted, or new rows can be added; however, no columns can be deleted. You can also create an entirely new known regions file and load it using the Filter Table interface. For more information about adjusting or creating new Known Regions lists, see the User Guide.
No, .cif files cannot be analyzed with BaseSpace. Additionally, it is not possible to output .cif files with HCS v2.2 on HiSeq v4 mode or Rapid Run mode with HiSeq v2 chemistry. The option to output .cif files is available in TruSeq v3 mode and Rapid Run mode with TruSeq chemistry.
If you are using CASAVA, you can analyze rapid run data with CASAVA. If the zip BCL files option was chosen during run set up, you will need to use the bcl2fastq converter in place of the configureBclToFastq component of CASAVA. For rapid runs, you will align data from each flow cell separately and then merge the data at the configureBuild step.
If you are not using CASAVA, note that Illumina is discontinuing distribution of CASAVA software to better support new products available on BaseSpace. BaseSpace features analysis options for a large array of NGS applications.
If you currently use CASAVA, you can analyze HiSeq v4 data with CASAVA. You need to use the bcl2fastq v1.8.4 conversion software in place of the configureBclToFastq component of CASAVA.
For HiSeq v4 runs, perform alignment of data from each separately, and then merge the data at the configureBuild step.
If you are not using CASAVA, note that Illumina is discontinuing distribution of CASAVA software to better support new products available on BaseSpace. BaseSpace features analysis options for a large array of NGS applications.
No, this is not an option in the PC Module.
Yes, when adjusting the analysis settings from a current analysis, a new analysis is initiated. Additionally, the alteration of sample indexes or sample types automatically initiates a new analysis.
As with any molecular biology techniques, a change in protocol will give a change in absolute results. We do not recommend comparing absolute counts (or counts normalized to total counts) between different preparation protocols. However, comparison of fold change between the two protocols should yield good correlation.
The data is comparable to TruSeq RNA Sample Prep v2 data. The TruSeq stranded RNA and Directional mRNA-Seq protocols are based on distinct ligation chemistries and the libraries they produce are expected to differ as a result. Direct sample to sample comparison is not recommended for these library types.
Illumina strongly advises against this, as each product contains a different SNP list. Instead, download samples from a single array type and version number in each session.
Yes. Direct sample to sample comparison can be performed using the ForenSeq Universal Analysis software.
Intensity (Data by cycle) plots appear different due to non-linear exposure ramping. Non-linear ramping prevents exposure damage early in the read, which provides a boost later in the read when it is more necessary.
There are variances between the TruSeq LT and HT kits that may result in differences in yield and the amount of adapter dimer for the final library. Be sure to take extra care pipetting the AMPure XP beads accurately in the Clean Up PCR step, to minimize the amount of adapter dimer carried through to the final library. Also, note that the AMPure XP bead ratio at this step is optimized for the longer length of the dual-index adapters.
Intensity for the G channel is expected to be lower, but the rate of decay is much slower due to one of the new reagents in the TruSeq SBS Kit v3 - HS called SRE. Therefore, your data quality will not be impacted.
You can export your data as multiple *.csv files or as a single Excel file with multiple worksheets. In addition, you can export a report as a PDF or PowerPoint file.
Yes, the adapter plate can undergo freeze/thawing up to 4 times. More freeze thaws of the plates may impair assay performance.
It is possible to use larger fragment sizes, however, we have found that shorter fragment sizes generate the best coverage. Please refer to Appendix A in the user guide for the TruSeq RNA sample prep kit that you are using for more information.
Since it is a new instrument that has not been released to market, there are currently no publications citing the use of the Eco system. Some data are available on www.ecoqpcr.com. Check back periodically and keep an eye on the monthly Illuminotes newsletter for updates.
Yes. VariantStudio v2.2 and v2.2 can be installed concurrently on the same computer. The two versions of VariantStudio operate independently; information is saved in different application spaces. VariantStudio v2.1 installs as "Illumina VariantStudio 2.1", while v2.1 installs as "Illumina VariantStudio 2.2". Therefore, VariantStudio v2.2 does not overwrite the v2.1 installation. Using the default installation folder for both versions guarantees concurrent usability. If you decide to define a custom installation path, make sure that you do not use the same installation folder for both versions.
The probe design and hybridization conditions were optimized for use with amplified cRNA. Varying the sample to cDNA would require re-optimization of every step of the protocol’s hybridization and wash conditions. Our protocol does not include a pre-block step of the microarray before adding sample. Adding Cy3-labeled sample would produce high background and poor results.
Yes. However, if analysis software other than Illumina analysis software is used to generate data, the VCF file might not contain the columns required by VariantStudio. See the VariantStudio User Guide (part # 15040890 B) for information about VCF file requirements.
Yes. You can import custom annotations to the software using the Custom Annotation function in VariantStudio. This feature requires that a custom annotation file uses a tab-delimited text format (TSV) and contains a header row that specifies the column names. VariantStudio allows both custom variant annotations and custom gene annotations.
No, it is not possible to generate a new project in the PC Module directly from idats. The polyploid workflow requires to first generate a genotyping project in the GenomeStudio Genotyping Module from the idats. The genotyping project (.bsc) can then be opened in the PC Module for polyploidy clustering.
Yes. By default, VariantStudio provides five classification categories: Pathogenic, Presumed Pathogenic, Unknown Significance, Likely Benign, and Benign. You can edit these categories through the Annotation & Classification | Classification Settings menu.
Illumina provides the Sequencing Analysis Viewer (SAV) software that can be run on a Windows PC to remotely monitor your run. The software does not allow any control over the run and requires that the PC is connected to the analysis server over the network. Another application you can use to monitor your run is SeqMonitor, which allows you to monitor your run using your iPhone or iPad.
Yes, the SNP list is available for download:
Yes, please use the following catalog numbers:
FT-260-1002 for Infinium OncoArray FastTrack Service
FT-260-1012 for Infinium OncoArray+ FastTrack Service (for the OncoArray plus add-on)
Yes. Please select the appropriate workflow based on the flow cell type (Single Read or Paired End) when starting your run so that the correct chemistry is used. If using a single-read flow cell the TruSeq Dual Index Sequencing Primer Box, Single Read (single use box) (catalog# FC-121-1003) is necessary in order to sequence both index reads.
No. Comparisons of two or more groups of samples cannot be performed using the application.
VariantStudio supports family-based filtering of father, mother, affected child, and affected or unaffected siblings. In VariantStudio v2.2, the analysis requires input of the proband and one other sample, either a parent or a sibling. This process filters variants that are consistent with a particular inheritance mode, including autosomal dominant, autosomal recessive, X-linked recessive, and de novo mutation.
Yes. Sample to sample comparison is done through the Cross Sample Subtraction Filter. This process filters variants that are present in one sample but not the other.
Real-Time Analysis (RTA) v1.18 includes optimizations to the algorithms that identify clusters and estimate the color normalization matrix and phasing and prephasing rates. These optimizations improve the ability of Real-Time Analysis to handle low-diversity samples, such as samples with unbalanced genome compositions (AT- or GC-rich genomes) or samples with low sequence diversity (amplicon sequencing). Because of these improvements, it is no longer necessary to designate a control lane in the control software to estimate matrix and phasing. For details, see Low-Diversity Sequencing on the Illumina HiSeq Platform.
You can install a second licensed copy of the MiSeq Reporter on another computer. However, that computer must be connected to the same network as your MiSeq or the network location of the output folder. The computer must meet the following minimum hardware and software requirements: 64-bit PC with at least 8GB RAM (16-32 GB RAM for optimal performance), Windows Vista or Windows 7, and at least 1 TB of available hard disk space.
Illumina does not support, and strongly advises against, running libraries prepared by different sample prep kits in the same lane of a flow cell. We do support running libraries prepared by different sample prep kits in different lanes of the same flow cell or spiking in llumina PhiX control library in the same lane as any user-prepared libraries. If different library types are run in different lanes, Dual Index Recipes and the Dual Index Primer Box must be used. The indices between TruSeq HT and Nextera are unique and not shared. Please see appropriate sample prep user guides for index sequences.
Yes. However, for optimal coverage, Illumina recommends sequencing stranded RNA libraries with paired-end chemistry.
Yes. You can run any library on the MiSeq provided your library has complementary adapters.
No, only runs of the same mode can be performed simultaneously. If you run TruSeq v3 mode on side A, then you must run TruSeq v3 mode on side B. The same is true for running HiSeq v4 mode. For Rapid Run mode, you can perform a rapid run on both sides using TruSeq Rapid kits on one side and HiSeq Rapid v2 kits on the other.
In HiSeq v4 mode .cif files cannot be saved. The option to save .cif files is available in other modes.
Yes. After selecting filter settings using options in the Filters pane, you can save the filter for later use. To apply a saved filter, click the drop-down menu in the Current field, and select a filter name from the list. The filter is automatically applied.
Yes, you can sequence your enriched libraries on the MiSeq System, obtaining results in hours instead of days. However, the MiSeq Reporter currently cannot process the manifest file provided for your enrichment experiment. Therefore, the MiSeq Reporter maps your reads to the entire genome, not just the enriched regions, and alignment of the reads takes at least four hours.
Prepare your libraries as described in the protocol for TruSeq Exome Enrichment Kit or the TruSeq Custom Enrichment Kit, and prepare your libraries for sequencing as described in the MiSeq System User Guide. When you are ready to set up your sample sheet using the Illumina Experiment Manager, select the following settings:
MiSeq Reporter will generate the standard Resequencing reports. Consequently, you will need to zoom in on your enriched regions to obtain relevant data.
A single side of the instrument can be switched from one mode to the other. However, after a run begins on one side, a run cannot be started on the other side unless it is the same mode. Perform mode switching procedures on the side that you intend to sequence on in the new mode. For example, if you have completed 2 rapid runs on side A and B, and want to set up only 1 high output flow cell on side A, change the mode for only side A.
A high output run on side B cannot be performed until a mode change is complete on side B. For efficiency and the most run flexibility, perform mode switching on both sides of the system at the same time.
The GoldenGate Assay can tolerate relatively short stretches of target DNA (> 200 bp) and can be quite tolerant of degraded FFPE samples. Internal experience with FFPE samples used with the GoldenGate Assay indicates that high-quality genotyping data can be obtained. Decreased call rates from FFPE samples compared to genomic DNA samples may be observed, but the decrease in call rate depends on the level of sample degradation.
No. HiSeq X flow cells are designed and optimized for use on the HiSeq X system. HiSeq X flow cells are not compatible with other HiSeq models.
No. HiSeq X reagents are designed and optimized for HiSeq X systems. HiSeq X reagents are not compatible with other HiSeq models.
No. MiSeq kits use an all-inclusive reagent cartridge system that is specifically designed to deliver the reagents necessary for cluster generation, sequencing, and paired-end chemistry.
No. The Illumina VariantStudio End User License Agreement (EULA) states that Illumina VariantStudio must be used solely to analyze data generated from an Illumina sequencing instrument.
No. File directory structures from a HiSeq system are incompatible with MiSeq Reporter software.
However, the TruSeq Amplicon App is available in BaseSpace and can be used to analyze the TruSeq Amplicon Cancer Panel, the TruSight Myeloid Sequencing Panel, and the TruSeq Custom Amplicon Panels.
No, file directory structures are incompatible with MiSeq Reporter software. However, the TruSeq Amplicon App is available in BaseSpace and can be used to analyze the TruSeq Amplicon Cancer Panel, the TruSight Myeloid Sequencing Panel, and the TruSeq Custom Amplicon panels.
Yes, NuPCR works on all instruments from major manufacturers. It has been shown to work on the following instruments:
Yes. SAV 1.8.37, or later, is required to view data from a HiSeq X run. You can download SAV for use on a networked computer or use SAV in BaseSpace.
Yes. SAV 1.8.36 is required to view data from a NextSeq run. You can download SAV for use on a networked computer or use SAV in BaseSpace. SAV 1.8.36 is also compatible with HiSeq and MiSeq run data.
No, kits are not interchangeable.
Yes, the low sample (LS) and high sample (HS) workflows may be applied with both the high throughput (HT) and low throughput (LT) kit configurations. Please refer to the user guide for the TruSeq library prep kit that you are using for Illumina's recommendations on kit and protocol combinations to ensure the best results.
Illumina does not recommend reusing plates as there is a significant risk of contamination.
Illumina recommends using a microheating system during tagmentation and enrichment wash steps. However, if a microheating system is not available, a thermal cycler can be used. Set up the Tagmentation reaction as instructed in the User Guide using a 96-well hard-shell plate. For the enrichment wash steps, the large sample volume requires adjustments to be made to the protocol for robust performance. Refer to Appendix A in the User Guide for complete details.
While it is possible to create libraries for sequencing on the Genome Analyzer from degraded RNA, it will have a large effect on the performance of the assay. One of the first steps in the process is the purification of poly-A mRNA using a poly-T capture step. If the RNA is degraded, the mRNA that is captured at this step will not be full length, and will not give full-length cDNA products after random priming. High-quality total RNA produces even, end-to-end coverage of each mRNA molecule. If the RNA is degraded, you might observe a noticeable 3'- to 5'-bias in the number of reads for most transcripts. Greater degradation will result in greater bias.
When you compare expression levels across many samples, compare counts only from samples that have a similar RIN (RNA integrity number) from the Agilent Bioanalyzer to give the most quantitative results.
No. The same sequencing primer is distributed across both lanes of a rapid flow cell as part of the HiSeq 2500 on-instrument cluster generation workflow.
The new HiSeq v4 reagent kits now support dual indexing workflows without requiring the purchase of additional SBS agents. Sample prep for dual-indexed libraries requires that both indexes be present on the library. However, the second index does not need to be read during sequencing. A single-indexing workflow is supported on Illumina sequencing instruments, where only Index 1 is used. See the instrument user guide for more information about setting up an 8-base single-indexed sequencing run.
All TruSeq DNA sample prep kits, including the HT version, supports input of standard genomic DNA only. Illumina offers other kits for Amplicon library prep. The TruSeq DNA HT Sample Prep kit supports high quality genomic DNA as input for sample prep to ensure best results. Please see the DNA Input Recommendations section of the TruSeq DNA Sample Preparation Guide for further information.
The adapter plate is single-use per well and Illumina recommends that it not undergo more than four freeze/thaw cycles. For this reason, Illumina strongly recommends processing at least 24 samples at a time with the TruSeq HT library can be pooled and sequenced in any Illumina supported configuration including low-plex. If it is desired to prepare only a few samples at a time, Illumina recommends purchasing a TruSeq LT library prep kit.
Yes, the modified cycling incubation can be used on the GoldenGate Methylation Assay. It is not required, however, because that assay is more tolerant towards incomplete conversion.
The MiSeq provides sufficient output to run a single Nextera Rapid Capture Enrichment per MiSeq run. However, the HiSeq 2500 continues to be the most cost effective and highest throughput platform for exome sequencing.
Yes, TruSeq DNA HT libraries can be used for downstream enrichment using either the TruSeq Exome Enrichment or TruSeq Custom Enrichment kits. There are slight differences in the design of the v2/LT and HT adapters; therefore, use of the HT adapters may result in slightly lower percent enrichment compared to the v2/LT adapters.
Yes, as long as the index read primer and multiplex read 2 primer are used. TruSeq DNA and RNA libraries have the same architecture and sequencing primer attachment sites as v2 multiplexed libraries.
Illumina does not support running libraries prepared by different sample prep kits in the same lane of a flow cell and does recommend running any combination of library types in a single lane. Please note that Illumina PhiX can be spiked in to any lane with user-prepared libraries.
No, not currently. The Directional mRNA-Seq Sample Preparation protocol would need to be optimized to take the longer adapters into account, as the size difference makes adapter dimers more difficult to distinguish from actual inserts using SPRI or column purifications. Illumina encourages customers to continue to use the Illumina Small RNA v1.5 adapters as specified in the Directional mRNA-Seq Sample Preparation Guide.
TruSeq adapters are methylated. However, the kits are not currently compatible for the following reasons:
Yes, the TruSight One Sequencing Panel is designed to allow trio sequencing and data analysis using the MiSeq system. The 9-sample TruSight One kit is intended for use with the MiSeq and contains three MiSeq v3 reagent cartridges. The 36-sample kit does not contain sequencing reagents and can be sequenced on any Illumina sequencer.
To log in to MiSeq FGx Control Software, a connection must be established with the ForenSeq Universal Analysis Software server to check authentication. After authentication, an RUO run can be performed without connection to the server. There is a buffering mechanism with the MiSeq FGx Control Software to allow data transmission to resume upon a connection loss to ForenSeq Universal Analsyis Software during acquisition.
Yes, the nucleotide distance of all the indices is such that mismatch of 1 still makes a unique index.
Yes. The MiSeq reagent cartridge includes three empty reservoirs for custom primers. You have the option of using a custom primer for Read 1, the Index 1 Read, and Read 2. For more information, see Using Custom Primers on the MiSeq (part # 15041638).
Yes. There are three reservoirs on the NextSeq reagent cartridge reserved for custom primers: #7 for a custom Read 1 primer, #8 for a custom Read 2 primer, and #9 for a custom Index 1 primer.
Degraded DNA generates libraries with the kit. Depending on the extent of degradation, not all of the large amplicons in the multiplex can be detected. The kit can also overcome high levels of inhibitors that can be present in forensic samples.
Illumina supports sequencing libraries prepared by different sample prep kits such as single-indexed libraries and dual-indexed libraries in different lanes of the same flow cell. To do this, you must use a dual-indexed workflow and the appropriate dual-indexed primers, and prepare two sample sheets. For more information, see Sequencing Mixed Libraries on a HiSeq or GA Flow Cell.
No, a polyploid project (.pcm) can only be opened using the PC Module.
For paired-end flow cells, only Index 1 can be re-hybed, and this must occur before Index 2 begins. For single-read flow cells, Index 1 and Index 2 can be rehybridized before the run completes.
The protocol is optimized for the inclusion of a gel size selection step selecting for a DNA insert of 150–200 bp. Longer inserts could be used, however, this may decrease the signal to noise for the binding motif and may require modification for optimal run performance. Illumina recommends selecting a gel size range of 250–300 bp for the final library.
If pooling fewer than the number of indices provided in the kit, it is necessary to consider low-plexity index combinations. Color balance during the index read is needed to ensure proper image registration. If there is no signal in one of the color channels (red or green) of the index read, image registration may fail and no base will be called for that cycle. If no base is called, the index read may not be able to be matched to the sequence specified in the sample sheet, and then samples will not be able to be demultiplexed. Refer to the TruSeq Library Prep Pooling Guide for recommendations and guidelines for Illumina sequencing systems that require balanced index combinations. The Illumina Experiment Manager (IEM) will give warnings when generating sample sheets if the index combinations do not meet this requirement.
Yes. Report templates created and saved in VariantStudio are available to other VariantStudio projects.
Coverage of GC regions can be impacted by the model, settings, and performance of the thermal cycler used. Illumina has validated the Bio-Rad DNA Engine Tetrad 2, the Bio-Rad S1000, and the MJ Research PTC-225 DNA Engine Tetrad thermal cyclers. Other thermal cyclers may differ in their performance across the genome.
Yes, TruSeq cluster kits are backwards-compatible.
No. Rapid run SBS reagents and high output SBS reagents cannot be combined. Each set of reagents has been formulated specifically for use only with its respective flow cell and instrument run parameters. The cluster kits and flow cells are also paired with respective kit types and cannot be combined.
Yes, samples can be analyzed in a single experiment. We have observed high concordance between detected probes using MCS3 versus MCS4 plus RTE and have found the variability in raw signal intensity correlations between MCS3 and MCS4 plus RTE to be comparable to the variability between freshly made MCS3 and MCS3 nearing the end of its shelf life. As is to be expected, signal intensity correlations and detected probe concordance decrease with input RNA quality.
The Relative Quantification application calculates and displays fold changes automatically if you defined your reference sample as well as your reference assays. The software automatically calculates and displays error bars for each data point on the graph.
No, there is currently no Small RNA v1.5 oligo-only kit available.
Not at this time. 5-hmC behavior is indistinguishable from standard 5-mC in the Infinium Assay.
Yes. The MiSeq FGx Control Software offers an RUO run option alongside the Forensic Genomics run options. The RUO side of the software is fully compatible with all Illumina-supported RUO run types, and runs are set up as for standard MiSeq runs. For more details on set-up and workflow, see the MiSeq FGx Instrument Reference Guide.
Yes, the same metrics that are shown during data acquisition are available on the ForenSeq Universal Analysis Software after the run is complete.
FASTQ files are generated during secondary analysis by MiSeq Reporter for most analysis workflows. To generate only FASTQ files, specify the GenerateFASTQ workflow in the sample sheet, which generates FASTQ files and then exits secondary analysis.
No, the PC Module performs cluster assignment, but does not call genotypes. This is because the assignment of genotypes polyploid species is highly dependent on the population and biology of the organism. Any downstream genotype assignment should be done with the biology and evolutionary history of the population taken into consideration.
The DSN Normalization protocol was optimized for pre-TruSeq libraries. The TruSeq RNA libraries are not readily amenable to the DSN treatment. There are two primary problems:
Yes. To run the protocol without the controls, substitute Resuspension Buffer (RSB) for the control mixture at each step. See the appropriate TruSeq DNA or RNA sample preparation guide for details.
Illumina does not recommend using the microTUBE AFA Fiber Screw-Cap tube (Covaris Part # 520096) with the TruSeq Nano DNA Library Prep protocol. The solution tends to stick to the sides of the tubes and to the two rods inside the tube, resulting with probable sample loss. Illumina has optimized the M220 shearing conditions using the microTUBE AFA Fiber Pre-Slit Snap-Cap (Covaris Part # 520045) tube and the microTUBE AFA Fiber Crimp-Cap tubes (Covaris Part # 520052).
The protocol is optimized for 18 cycles of PCR to allow for variability in sample input amounts and quality. High quality samples will likely be able to use a lower number of PCR cycles and still generated adequate amounts of final library. A titration of PCR cycles can be performed to identify the optimal performance for particular samples. Illumina does not recommending increasing the number of PCR cycles as it may skew the representation of the library.
No. However, a sample sheet created using IEM is compatible with the RUO mode of the MiSeq FGx Control Software. For forensic runs, the samples are defined in the ForenSeq Universal Analysis Software before acquisition through a file upload or manual entry. For details on the information in the file, see the ForenSeq Universal Analsyis Software Guide.
No, the wells cannot be reused.
Yes. Please note that we have seen high sample variability using some commercially available small RNA spin column purification methods, and validation of any purification method may be necessary. If purified small RNA is used as starting material, 10–50 ng should be used.
No, variant classification and associated notes saved in the Classification Database are not versioned. If the classification category or notes are modified for a given variant in the Classification Database, previous information is overwritten with the current modification.
Currently, batch processing of multiple Eco systems is not supported. Check back periodically and keep an eye on the monthly Illuminotes newsletter for updates.
On average, there are several hundred long-read molecules per well. Individual wells produce many long read fragments randomly sampled from across the genome.
Yes, in order for the results to be transferred from the MiSeq FGx to the ForenSeq Universal Analysis Software a connection must exist between the systems. In many cases that connection is established through the company network. However, in some labs that connection can be on a local network created with the provided router.
Yes, the MiSeq FGx needs must be washed between runs to remove any remaining reagents from the fluidics lines and make sure that they are clean before the next run is started. The MiSeq FGx offers a bleach post-run wash protocol that minimizes run-to-run contamination. See the MiSeq FGx Instrument Reference Guide for wash instructions.
Yes, a cBot is needed for cluster generation on any 8-lane high output flow cell, including HiSeq v4 and TruSeq v3.
You can transfer sequencing data from a NextSeq run to BaseSpace for analysis and storage. Additionally, you can configure the NextSeq to transfer data to a local server and perform analysis using third-party software.
Yes. To convert zipped .bcl files, use bcl2fastq v1.8.4. This version can also convert non-zipped .bcl files.
Yes, you need to supply an approval letter from your Institutional Review Board (IRB) or equivalent review committee (for international customers) before you begin the upload process. Please see the iControlDB support documents for additional information about the IRB documentation required.
If sequencing TruSeq HT libraries with the HiSeq, HiScanSQ, or GAIIx system on single-read flow cells, the TruSeq Dual Index Sequencing Primer Box, Single Read (single use box) (catalog # FC-121-1003) will need to be ordered separately when using both indices. This add-on box is not required if sequencing a TruSeq HT prepared library with the MiSeq System or on paired-end flow cells for HiSeq, HiScanSQ, GAIIx. Additional SBS reagents may be necessary to account for all index cycles.
No, you can use different algorithms and clustering options for different SNPs. The goal is to find optimal parameters for each SNP matching the biology of your samples.
Yes. Priming SBS reagents is required. The control software prompts for this step, which takes about 15 minutes.
No. Priming is integrated into the HCS workflow for Rapid Run mode.
No, TruSeq DNA PCR-Free library prep kits contain Sample Purification Beads (SPB) that are used for size selection and clean-up steps. They do not require the user to supply AMPure beads separately.
No, the kit contains Sample Purification Beads (SPB) that are used for size selection and clean-up steps. They do not require the user to supply AMPure beads separately.
The TruSeq Dual Index Sequencing Primer Kit (catalog #PE-121-1003) is required for clustering libraries on the cBot and paired-end sequencing on the HiSeq 2000, GAIIx, and in High Output mode on the HiSeq 2500. This kit is good for one run and contains the required primers for dual index sequencing (HP10, HP11, HP12).
The dual index primers (HP10, HP11, HP12) are included in MiSeq reagent kits and HiSeq 2500 Rapid reagent kits and do not need to be purchased separately for these types of runs.
Yes. The new cluster protocol and wider lanes on flow cell v3 make it imperative to re-titrate your samples to optimize cluster densities for higher throughput. qPCR is highly recommended.
No reference genome is necessary for the MiSeq metagenomics workflow.
No. Cluster generation is performed on the MiSeq instrument as part of the run. No other instruments are required for sequencing on the MiSeq.
Final libraries are single stranded and cannot be quantified. Follow the normalization and pooling protocol before loading onto the MiSeq FGx.
The project scientist that is assigned to you works with you to submit representative test samples. These samples are quantitated at Illumina using PicoGreen to confirm sample concentration. Samples are genotyped on an Illumina array to determine genotyping quality. This data set is not for distribution. The data set is used exclusively for sample quality determination. After successful quality determination, the Illumina project scientist works with you to submit the production samples.
Yes. Illumina FastTrack Sequencing Services offer a human phasing sequence analysis service. See the Phasing Analysis Service FAQ for more details.
Yes. Illumina FastTrack Services offer a cancer analysis service for tumor-normal data set comparison. The standard offering is for a 40x normal and 80x tumor pair. See the Sequencing Service Process for more information.
The sequencing team performs downstream analysis for the cancer analysis service. For tertiary data analysis of standard whole-genome sequencing services, the Illumina Genome Network works with software partners who are experts in the field.
No. Dual-indexed libraries sequence eight bases of actual index sequence rather than six bases plus one more for phasing calculations. For short sequences there are no phasing/prephasing corrections, so it is not necessary to sequence one additional cycle.
TruSeq Stranded Total RNA Sample Prep kits remove ribosomal RNA only. The Human/Mouse/Rat kits remove cytoplasmic ribosomal RNA and the Ribo-Zero Gold kits remove cytoplasmic plus mitochondrial ribosomal RNA.
No testing has been performed on the effects of local proxies on BaseSpace access.
If you choose to use BaseSpace only for run monitoring and your samples are not indexed, a sample sheet is not required. If you want to use BaseSpace for data storage and analysis, a sample sheet is required. The sample sheet can be in either HiSeq Analysis Software format or CASAVA format. When using BaseSpace, combining indexed and non-indexed samples on a flow cell is not possible.
Yes. The Amplicon-DS workflow in the MiSeq Reporter software is used for the alignment and variant calling of TruSight Tumor data. The resulting VCF files can be imported into VariantStudio. Illumina recommends that you use the merged VCF file from the Amplicon-DS workflow. In these files, the variant call information from both the forward pool and reverse pool has been merged.
Yes. VariantStudio v2.1 and v2.2 support VCF and gVCF file formats. For more information, see the VariantStudio User Guide.
No. Illumina provides annotations from the respective annotation sources as is.
Illumina does not provide any recommendations as methods for ChIP pulldown and fragmentation are dependent upon individual antibodies and procedures. Please reference literature or other sources for recommendations.
Oligo Only Kits are not offered for the TruSeq DNA and RNA Library Prep kits.
Yes. Illumina offers a TruSeq Custom Enrichment kit. Reference the TruSeq Custom Enrichment Data Sheet for more information.
No, Illumina does not provide antibodies.
No, Illumina does not provide any classified variants. VariantStudio provides an empty Classification Database that you can populate by uploading classified variants from an external source or by manually classifying variants in samples that are being analyzed in VariantStudio.
Illumina has not extensively tested any of the commercially available standards, and does not recommend any of them at this time. In house, Illumina has used standards generated in our own laboratory. Our methods for generating the standards are described in the following article:
Bibikova M, Le J, Barnes B, Saedinia-Melnyk S, Zhou L, et al. (2009) Genome-wide DNA methylation profiling using Infinium assay, Epigenomics 1:177-200
Background subtraction is required when you compare data run on different types of scanners because of technical disparities: for example, the iScan and HiScan have very different offsets. Background subtraction has a much smaller effect when you scan chips on the same scanner, and might not be necessary. We suggest that you analyze a subset of data with and without subtraction, and choose the one you prefer based on your results.
No. Filtering parameters vary depending on the application and objective of the investigator.
Requantitation is not necessary as long as you use at least 500–1000 ng DNA for the bisulfite conversion. It is critical to accurately quantitate the input DNA concentration with PicoGreen to ensure that you add sufficient DNA to the bisulfite conversion reaction. Bisulfite conversion renders DNA less complementary, so much of the DNA will be denatured and be more difficult to quantitate accurately.
Yes. Illumina recommends the following of-the-shelf bisulfite conversion kits from Zymo Research.
Catalog NumberD5001 50 reactions (single-column format)
D5002 200 reactions (single-column format)
D5004 2×96 reactions EZ-96 DNA methylation kit (deep-well format)
Automation of the DASL® Assay is available. However, Illumina currently does not offer automation for the Direct Hyb protocol. Contact Ambion for information about automation of the TotalPrep protocol.
The use of a UPS is optional. However, a UPS is highly recommended to protect the instrument in the case of a power surge. For more information, see the MiSeq System Site Prep Guide.
In the random priming process to generate cDNA with the best coverage, this information is not retained. If originating strand information is required, please refer to the Directional mRNA-Seq Sample Preparation Guide or other published applications.
No, you perform variant classification. With VariantStudio, you can provide annotations that can be used to classify variants within VariantStudio. After you assign a classification category to a variant, the information is saved to the Classification Database so that it can be applied to the same variant observed in other samples. Classifications saved in the Classification Database can be applied automatically to other samples through the Apply Classifications from Database menu.
Yes, Illumina VariantStudio requires internet connection for annotating variants because the annotation database, known as the Illumina Annotation Service, resides in Amazon Web Services. After variants are annotated and saved in a project, an internet connection is not necessary.
No. VariantStudio is tested and supported on Windows 7 or later. Virtualization software like Parallels or VMware lets you run a Windows application on a Mac or Linux. Therefore, it is possible to run Illumina VariantStudio through Parallels on a Mac. However, this method is an unsupported function. It is recommended that you give your VM at least 2 GB of RAM and close any other programs.
cDNA libraries should be considered an equivalent to gDNA for clustering and sequencing steps, assuming an equivalent complexity of the sample.
No. In addition to instrument control and data collection capabilities, the Eco Real-Time PCR System comes with the EcoStudy data analysis software. If more advanced analysis is required, you can export all raw data in Excel or CSV format for convenient analysis in other third-party software.
No, the HiSeq Analysis Software provides analysis of libraries prepared with the Nextera Rapid Capture exome enrichment kit and human Whole Genome Sequencing using only the hg19 genome as a reference. CASAVA provides analysis of additional applications such as RNA sequencing, Exome sequencing, targeted resequencing, and Whole Genome Sequencing using a more extended set of reference genomes available on Illumina.com's iGenomes page. (http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn). More information on CASAVA is available here: http://www.illumina.com/software/genome_analyzer_software.ilmn#casava
Yes. The MiSeq includes a fully-integrated instrument computer that runs on Windows 7 Embedded and contains the operating software, control software, and analysis software.
The Ribo-Zero Globin kit supports human, mouse, and rat. Use of this kits with other organisms has not been tested and is not supported.
Please contact Illumina technical support with the rRNA sequence for your organism of choice to determine if your organism is compatible with the Ribo-Zero Globin kit. Please note that the analysis will be done in silico and does not guarantee rRNA removal.
The Ribo-Zero Plant kit will remove rRNA from a broad variety of plant RNA samples. It has been laboratory tested against Arabidopsis, maize, wheat, rice, corn, and soy bean. In silico testing, it has also demonstrated compatibility with Japanese honeysuckle (Lonicera japonica), mirror bush (Coprosma repens), moss (Physcomitrella), Tomato (Solanum lycopersicum), Vietnamese coriander (Persicaria odorata), and water clover (Marsilea vistita).
To check whether your organism of choice is compatible with this kit, see the Will a TruSeq Stranded Total RNA kit work with my organism? bulletin. If your organism of choice is not listed, use RNAMatchMaker to find which Ribo-Zero kit is compatible. Note that the analysis will be done in silico and does not guarantee rRNA removal.
The acquisition is done by a CCD camera through the top of the plate and through the optical adhesive sealer.
Use the Eco evaluation plate to test the performance of the instrument whenever needed.
The files uploaded as instrument health data are RunInfo.xml, RunParameters.xml, RTAComplete.txt, InterOp files, and RTAConfiguration.xml.
Dual-indexed runs on the HiSeq comprise 8 bp of index sequence rather than 6 bp plus a seventh for phasing calculations. For more information, see the user guide for your sequencing instrument.
Please reference the Illumina iControlDB web page http://www.illumina.com/science/icontroldb.ilmn and/or Illumina, Inc., San Diego, CA, whichever is the preferred format of the journal to which you are submitting.
No, the tile numbering is unchanged from the format introduced in HCS v1.3. However, when using Flow Cell v3, the tile numbering reflects the three-swath imaging pattern, where a 3 in the tile number represents the third swath.
Yes. Our probes (human and mouse) have been mapped to Ensembl. The Illumina probe track can be turned on by checking the box next to Illumina Probes on the DAS Sources dropdown menu in Contigview. When the page refreshes, blue features indicate the location of probes. Clicking on a probe opens up a floating menu with probe information and a link to a page giving more information about the probe. The GFF text files giving the mapping information for probes to mouse and human can be downloaded from the Sanger Institute website ( http://www.sanger.ac.uk/Software/ formats/GFF/): - Human GFF file (right-click and select Save As) - Mouse GFF file (right-click and select Save As) More information about the GFF data format with an explanation of the fields can be found at: http://www.sanger.ac.uk/Software/formats/GFF/
If a new version of the Illumina Annotation Service is released at the same time as a new software version release, the updates to the annotation database are specified in the software release notes. Otherwise, you receive an email notification informing you that a new database version is available. The email outlines the changes made relative to the previous annotation database version.
ChIP-Seq runs should be aligned using the eland extended module of Gerald in CASAVA1.7. CASAVA 1.8 is not compatible with ChIP-Seq analysis. In CASAVA1.7, the Gerald config.txt file should include the line "WITH_SORTED true" to generate the sorted.txt files for each lane. CASAVA itself does not need to be run for ChIP-Seq runs. The GenomeStudio ChIP-Seq module requires the sorted.txt files and Summary.htm from Gerald.
A beta value (β) is calculated for each CpG locus.
Each kit includes two flow cells and all required reagents, including clustering, SBS, paired-end, and indexing reagents. One kit provides consumables for a 150-cycle paired-end (2 x 150 bp) sequencing run plus indexing on both flow cell A and flow cell B.
With the release of the HiSeq X Reagent Kit v2, a new kit configuration called the 10-pack kit is available. The 10-pack kit includes flow cells and consumables for 10 dual flow cell runs or 20 single flow cell runs. The 10-pack is designed to support the preparation and sequencing of 4 flow cells at a time, and significantly reduces storage requirements.
p = 10'(DiffScore*sgn(µcond-µref)/10)
Do the following to ensure that your network is performing fast enough for a particular run:
6. Repeat steps 2-5 for a few more files. The typical copy speed should be at least 10 Mbyte/s, ideally 15 Mbyte/s or higher.
7. If the copy speed is consistently significantly less than 10 Mbyte/s or lower, your network does not perform fast enough.
See the FAQ “What should I do if the copy speed is too slow?” for suggested solutions.
This situation is a challenge for any system trying to quantify gene expression levels. Variable quality is more challenging than variable compensations for the mRNA-Seq Assay. As described in the question about RNA degradation, we recommend against comparing results across samples that have a large range of quality and integrity, because there will be a large effect on the read coverage across the full length of the transcripts.
If your RNA samples are of distinctly different quality, start by re-purifying all of the lesser-quality samples with the goal of having the highest quality RNA possible. Next, check that the samples you want to compare are of similar quality and quantity. We do not yet have analytical methods to normalize for these effects in mRNA-Seq data.
To create a report for all the analysis results in a project, a report can be generated from the project page. To create a report for a sample result, an action is available from the sample details page to create the report. After a report is created, it can always be retrieved again from the project dashboard.
The sidebar in any of the Project screens contains all of the analyses with sample related to the selected project. Each analysis is individually selectable and immutable.
Hard copies of the setup poster and user guide are included with each Eco system, and a soft copy is available on the USB drive. You can also download PDFs of the Eco documentation from http://www.illumina.com/ecoqpcr.
Illumina will handle all service and repair requests. If you have a service or repair request, please contact Technical Support.
Amplification in NTC reactions can either be from contamination or non-specific amplification. Performing melt curve analysis can help identify if the signal is from contamination or from non-specific amplification.
If it is contamination, the melting curve of the NTC reaction will have the same Tm as your target sequence. Good aseptic technique, using aerosol-resistant pipette tips and a Real-Time PCR master mix with dUTP and UDG, can help to reduce any potential contamination.
If the signal is due to non-specific amplification, the melting curve of the NTC reaction will have a different Tm than the target sequence. The most common type of non-specific amplification is primer-dimer formation, and there are a number of ways to reduce this.
Optimal primer design is an important first step in preventing primer-dimer formation.
If these steps do not help reduce primer-dimer formation, redesigning the primers is necessary to obtain good results.
Data can be visualized for run monitoring within MiSeq FGx Control Software. ForenSeq Universal Analysis Software can be used to monitor runs and visualize the results of analyses.
You can zoom into a specific region of interest by clicking on the specific aberration that is shown within the Found Regions table. After this is done, a closer view of the aberration of interest is shown in the chromosome viewer.Alternately, use the zoom functions available in the toolbar or drag and stretch the red box on the ideogram to zoom in for a closer view of your data.
To determine the chromosomal location for the potential/putative miRNAs, Illumina used the following procedure:
When multiple 100% hits are found in the genome for one particular mature miRNA sequence, the chromosomal coordinates are listed by the score, i.e., the location with the best score is listed first.
Data analysis is performed using the ForenSeq Universal Analysis Software.
The system is designed to support multiple workflows inclusive of data analysis, which is performed on-instrument upon completion of the run. Output file formats are *.bcl, FASTQ, BAM, *.vcf, *.csv, and *.txt.
Nextera Rapid Capture Enrichment data can be analyzed using the HiSeq Analysis Software enrichment workflow for HiSeq data or MiSeq Reporter software for MiSeq data. See the HiSeq Analysis Software or MiSeq Reporter support pages for more information.
Alternately, Illumina sequence base call output files (*.bcl) can be demultiplexed and converted to FASTQ format using the bcl2fastq converter software. The files can then be used for analysis with other third party software packages (e.g., BWA and GATK).
Illumina suggests using a third-party methylation analysis solution such as Bismark, BSMap, or BS Seeker. See the Methylation Software section for more details.
RNA quality is perhaps the most important factor in generating reliable and reproducible Real-Time PCR data. Traditionally, RNA quality was assessed using gel electrophoresis and comparing the 28S and 18S ribosomal RNA bands. Gel electrophoresis is a laborious, time consuming, and low-throughput method that requires fairly large amounts of RNA.
Automated lab-on-chip capillary electrophoresis systems such as the Bioanalyzer (Agilent) and Experion (BioRad) have become popular tools for determining RNA quality. These systems use microfluidic technology to perform electrophoresis on glass chips at a miniaturized scale that overcome some of the issues of traditional electrophoresis. Data are presented as an electrophoretic trace of the RNA sample. The Agilent Bioanalyzer provides a quantitative measure of RNA integrity known as the RNA Integrity Number (RIN). A proprietary software algorithm examines the entire electrophoretic trace to determine RNA degradation and gives a numerical value between 1 and 10 to indicate RNA quality. An RNA sample with a RIN value of 10 is considered a highly intact sample where as a sample with a RIN value of 1 is considered a highly degraded sample.
The Eco sample loading dock comes with two plate adaptors. These plate adaptors have the same footprint as standard 96-well plates and can be used in standard centrifuge plate holders. Use both adapters to balance the centrifuge.
Selection of a reference gene or genes is a critical step for expression analysis using Real-Time PCR. Validation of reference genes for each experimental condition is critical for obtaining accurate Real-Time PCR data. Validation requires determining if expression of the reference gene is stable between cells of different tissues and if any experimental treatment affects expression.
The following paper contains a good summary of the validation process:
Dheda K., et al. Validation of housekeeping genes for normalizing RNA expression in Real-Time PCR. Biotechniques 2004; 37: 112-119.
The process starts with extracting and quantifying RNA samples from the samples under investigation (diseased vs. normal; treated vs. untreated). The next step is normalizing the input of RNA into the reverse transcription reaction. The expression of a panel of different reference genes is then measured by Real-Time PCR and the differences in Cq across the different samples is determined for each gene.
There are a number of software programs available for selecting appropriate reference genes, including GeNorm (http://medgen.ugent.be/~jvdesomp/genorm/), BestKeeper (http://www.gene-quantification.de/BestKeeper-1.zip) and Norm-Finder (http://www.mdl.dk/Files/NormFinder_0953.xla).
Illumina uses a green laser to sequence G/T bases and a red laser to sequence A/C bases. At each cycle at least one of two nucleotides for each color channel need to be read to ensure proper registration. It is important to maintain color balance for each base of the index read being sequenced, otherwise index read sequencing could fail due to registration failure. For pooling strategies for a small number of samples, please refer to the "Adapter Tube Pooling Guidelines" section in sample prep guide for the kit you are using . Additionally it is recommended to create a sample sheet in the Illumina Experiment Manager (IEM) prior to performing sample prep in order to confirm appropriate index combinations not listed.
Choosing which primer mix to use depends on the application and legislation in a particular country. DNA Primer Mix A can be used for casework and database applications. Due to legislation in some countries, ancestral and phenotypic data cannot be stored in the database and DNA Primer Mix B can then only be used for casework.
The fastest way to answer most questions is to refer to the Eco Support web page or in these FAQs. If you do not find an answer there, you can reach Technical Support by email at firstname.lastname@example.org, by phone at 1.800.809.4566 in the U.S., or on the Illumina website at http://www.illumina.com/support.
You can specify the GenerateFASTQ workflow in your sample sheet, which creates FASTQ files and then exits secondary analysis. For more information, see the MiSeq Sample Sheet Quick Reference Guide.
There are numerous primer design tools commercially available for purchase or freely accessible via the web. These tools simplify assay design significantly.
Primers and probes for NuPCR, DBD, and HRM assays can be designed using Illumina's the web-based DesignStudio software (www.illumina.com/designstudio).
Other primer design tools are Primer Express (Applied Biosystems), Beacon Designer (Premier Biosoft) and Real Time Design(BioSearch Technologies). Numerous websites contain databases of validated primer sets, including RTPrimerDB (http://medgen.ugent.be/rtprimerdb/) and the Quantitative PCR Primer Database (http://web.ncifcrf.gov/rtp/gel/primerdb/)
If you design primers and probes manually, follow these criteria from the Primer Express manual:
Designating dual indices on the sample sheet depends on whether a MiSeq or CASAVA sample sheet will be used. For MiSeq sample sheets, each index is entered into its own column. For CASAVA sample sheets, the indices are input in the format of "Index1-Index2". Please see the MiSeq Sample Sheet Quick Reference Guide or CASAVA User Guide for more information.
Illumina recommends that you use the Illumina Experiment Manager (IEM) to generate your sample sheet. The appropriate index format is automatically entered based on the selected sample sheet type.
In Real-Time RT-qPCR, genomic DNA can potentially be co-amplified during the PCR reaction, contaminating the sample and leading to erroneous results. To determine if an RNA sample is contaminated with genomic DNA it is important to include a no-reverse transcriptase control during the RT step, and all RT-qPCR experiments should include a no-RT control. If the RNA sample is free of genomic DNA contamination the no-RT controls should not generate any signal after Real-Time PCR.
To avoid genomic DNA contamination, treat RNA samples with DNAse before reverse transcription. Alternatively, design the PCR primers to anneal to sequences of the transcript that span a large intron. Primers designed in this way can only amplify cDNA.
The software provides a pass or fail indication for each positive and negative control sample. The pass/fail indication is based on the expected performance of the control.
The simplest and most commonly used method is the dilution or standard curve method. This method calculates PCR efficiency using the linear regression slope of a dilution series based on either of the following equations:
E = 10(-1/slope) -1
E = 10(-1/slope)The ideal slope is -3.32, which correlates to an amplification efficiency of 100%, meaning exactly one copy per cycle. Slopes in the range of -3.60 to -3.10 are generally considered acceptable for Real-Time PCR. These slope values correlate to amplification efficiencies between 90% (1.9) and 110% (2.1).
The Nextera XT DNA Sample Preparation Kit protocol is optimized for 1 ng of input DNA total. Illumina strongly recommends quantifying the starting genomic material. Nextera XT DNA Sample Preparation library preps use an enzymatic DNA fragmentation step and thus can be more sensitive to DNA input compared to mechanical fragmentation methods. The ultimate success of the assay strongly depends on using an accurately quantified amount of input DNA library. Therefore, the correct quantitation of the DNA library is essential.
To obtain an accurate quantification of the DNA library, it is recommended to quantify the starting DNA library using a fluorometric based method specific for duplex DNA such as the Qubit dsDNA BR Assay system. Illumina recommends using 2 μl of each DNA sample with 198 μl of the Qubit working solution for sample quantification. Methods that measure total nucleic acid content (e.g. nanodrop or other UV absorbance methods) should be avoided because common contaminants such as ssDNA, RNA and oligos are not substrates for the Nextera XT assay.
When using BaseSpace, sample sheet format can follow either HiSeq Analysis format or CASAVA format. For runs that require demultiplexing with either bcl2fastq 1.8.4 or CASAVA, a CASAVA-formatted sample sheet is required. This format is described in the bcl2fastq 1.8.4 User Guide (part # 15038058) and the CASAVA User Guide (part # 15011196).
Sample sheets for rapid runs include information for two lanes, as compared to eight lanes included in a sample sheet for a high output run. Sample sheets for rapid runs can be generated manually, using Excel or a text editor.
If you are using BaseSpace for data storage and analysis, a sample sheet is required for both rapid runs and high output runs. If using BaseSpace only for run monitoring and you are not indexing, a sample sheet is not required.
The BaseSpace Broker is designed to upload data to BaseSpace as soon as the data are generated on the HiSeq local drive. It will use as much bandwidth as is necessary to keep up with the data being produced. Under typical HiSeq run conditions, the upload of run data for storage and analysis will average less than 10Mbit/sec.
In most cases, throttling of the BaseSpace Broker data upload is not necessary. Throttling can be necessary if greater control over network bandwidth usage is required, such as sites where instruments share the network with other users or sites with limited upload speed. Throttling might be necessary in scenarios where the local network connectivity is temporarily lost and then restored. This interruption causes the BaseSpace Broker to suddenly consume more network bandwidth as it attempts to catch up with transfer of accumulated data. If no throttling is applied in such cases, the BaseSpace Broker might consume all available bandwidth on the network until the backlog of data are cleared. If throttling is applied and if the local network allows, Illumina recommends throttling to higher than the 10 Mbit/sec minimum specification. A recommended value of 20 Mbit/sec (approximately 3Mbytes/sec = 24Mbits/sec) allows the BaseSpace Broker enough bandwidth to recover, even if some delays in data transfer occur.
If throttling is needed, provide the following instructions to your local IT administrator:
Throttling of BaseSpace is performed on the HiSeq computer by application, rather than by IP address, as follows:
The run has met the Illumina specifications when the run metrics are within the thresholds and are colored green.
Using CASAVA: To merge data from different flow cells (different runs), use the configureBuild script in CASAVA v1.8.2. First, align the data (samples) from each flow cell separately using configureAlignment. Then, include each sample directory as an input directory in the configureBuild.pl command line. Input directories are specified by the -id option, as detailed on page 100 of the CASAVA v1.8.2 User Guide (Rev C).
If you are using CASAVA, note that Illumina is discontinuing distribution of CASAVA software to better support new products available on BaseSpace. BaseSpace features analysis options for a large array of NGS applications.
Using BaseSpace: BaseSpace includes a Sample Merge function that allows you to merge data from a single sample originating from different flow cells. This merging is performed before alignment analysis of the sample data.
The number of PCR cycles should be minimized to avoid skewing the representation of the library. Illumina recommends 15 cycles of PCR, which has been shown to provide the best performance in terms of coverage, reproducibility, and quantity of material. Although not recommended by Illumina, if you want to eliminate PCR, begin with a large amount of starting material (~5 μg) to generate equivalent yield as the standard protocol. Note that at least one cycle of PCR is required to open the forked adapters.
The TruSeq Custom Amplicon Kit uses a highly multiplexed assay to generate up to 384 amplicons across many samples with integrated barcodes for pooling prior to sequencing on a MiSeq. The Nextera XT DNA Sample Prep Kit can be used to prepare user-generated amplicons with standard PCR.
The TruSeq DNA PCR-Free protocol does not contain a PCR enrichment step to select for completed ligation products, therefore quantitation must be done by qPCR as other methods will quantify fragments that may not have adapters at both ends and will thus not generate clusters or sequence. See the Consumables and Equipment section of the TruSeq DNA PCR-Free Library Prep Guide for the required KAPA Library Quantification Kit. Follow qPCR instructions included in the KAPA Library Quantification Kits for Illumina sequencing platforms Technical Data Sheet using the KAPA standard, with the modifications specified in the Validate Library sections of the TruSeq DNA PCR-Free Library Prep Guide.
The most widely used method to quantify RNA is traditional UV spectroscopy. A diluted RNA sample is quantified by measuring its absorbance at 260 nm and 280 nm. The concentration is calculated using the equation:
[RNA] μg/ml = A260 x dilution factor x 40
where 40 is the average extinction coefficient for RNA
In addition, the A260/A280 ratio can be used to estimate RNA purity. An A260/A280 ratio between 1.8 and 2.1 indicates a highly pure RNA sample.
UV spectroscopy is relatively simple to perform but has several drawbacks. It does not discriminate between RNA and DNA so it is advisable to DNAse treat RNA samples before quantifying. DNA in the sample will lead to an overestimation of RNA concentration. Since proteins and residual phenol from the purification can interfere with absorbance readings, it is important to remove these contaminants in purification. Also, absorbance readings are dependent on pH and ionic strength. Dilute RNA samples in TE (pH 8.0) and use TE to blank the spectrophotometer before taking absorbance readings.
An alternative method for quantifying RNA samples is to use fluorescent dyes such as RiboGreen (Invitrogen). RiboGreen exhibits a strong fluorescent signal when bound to nucleic acids. Samples are quantified in a fluorescence microplate reader or standard spectrophotometer relative to a nucleic acid standard curve of known concentration. The linear range of quantification using RiboGreen is three orders of magnitude, from 1 μg/ml down to 1 ng/ml. The major advantage of fluorescent dyes over absorbence-based methods is that it is not affected by contaminating proteins or organic solvents carried over from the purification process. DNAse treatment is still recommended as RiboGreen does not discriminate between RNA and DNA.
RTA 1.9 (on GA) or 1.10 (on HiSeq) or higher processes the controls. To visualize them, SAV (Sequencing Analysis Viewer) 1.7 or higher must be used. RTA produces a new InterOp metrics file called ControlMetrics; this is the file SAV uses to get control counts.
MiSeq Reporter needs to have access to the repository, which is the location of the folder containing data for analysis. You can set this location in the settings window on the MiSeq Reporter main screen. When MiSeq Reporter has access to the repository, your runs appear in the Analyses tab in MiSeq Reporter. Select the Requeue checkbox next to the run you want to analyze, and then click Requeue to start analysis.
In order to perform dual-index sequencing in HCS 1.5, select the TruSeq Dual Index Sequencing Primer Box from the Index chemistry drop down menu on the recipe screen. This selection enables the use of the required chemistry for sequencing dual-indexed libraries, and must be used for sequencing any dual-indexed libraries (Nextera or TruSeq HT) regardless of which sequencing primers you will use for your run. Selecting any other setting will result in less than an eight-cycle index read.
Run data can only be uploaded to BaseSpace if the BaseSpace option is selected during run setup in the HiSeq Control Software. See the HiSeq 2500 System User Guide (part # 15035786) for information on setting up a run with a connection to BaseSpace.
For more information on BaseSpace, or to set up a free BaseSpace account, see https://basespace.illumina.com/home/index.
There are two methods for setting up non-serial dilutions or dilutions with a factor greater than 10. The following list shows a set of non-serial dilutions:
Automated method: Click the Standards button beside your standard assay to open the Set Up Standards pane, and then click Define Standards to open the Dilutions dialog box. Enter "4" for the number of points, 4450000 as the starting quantity, and 10 as the dilution factor. Back in the main Set Up Standards pane, directly type the correct values for the last two dilutions into the appropriate fields.
Manual method: Click the Standards button beside your standard assay to open the Set Up Standards pane. Type 4450000 directly into the first field, and then press Enter to make the next field active. Type the second quantity into that field and continue until you have entered all four quantities.
The following list shows a set of serial dilutions:
To enter serial dilutions with a dilution factor between 2 and 10, click the Standards button beside your standard assay to open the Set Up Standards pane. Click Define Standards to set up the serial dilutions.
Use the Enrichment workflow to create a sample sheet formatted for analysis of TruSight One data. Refer to the IEM TruSight One or TruSight Rapid Capture Quick Reference Card for instructions on creating your sample sheet.
If you are interested in a project with Illumina Fast Track Microarray Services, contact your sales representative or get a quote through the Illumina website. An Illumina sales representative discusses your genotyping project and helps you select the product that best fit your needs. The sales representative also provides a quote. After you receive the quote, a project manager contacts you to discuss your microarray project and the sample submission process. For both test and production samples, the Illumina project manager ships you a thermal cooler that contains barcoded plates, sealing mats, and return labels. If necessary, a courier picks up your samples. There is no charge for shipping.
If you are interested in a project with Illumina FastTrack Sequencing Services, contact your sales representative or get a quote through the Illumina website. An Illumina sales representative discusses your sequencing project and helps you to select the product that best fits your needs. The sales representative also provides a quote. After you receive the quote, a project manager contacts you to discuss your sequencing project and the sample submission process.
Use the DesignStudio table Export function to save designed regions, targets, and probes in .bed file format. Exported BED files can be imported into a genome browser, such as the Broad Institute Integrative Genome Viewer (IGV), which can be downloaded and run on your local PC computer, or the online UCSC Genome Browser.
Nextera sample preparation kits are used to prepare DNA samples for next‐generation sequencing. These kits use in vitro transposition to prepare sequencer‐ready libraries from genomic DNA for all Illumina sequencing platforms. The technology simultaneously fragments and tags DNA in a single tube reaction and this process is referred to as "tagmentation". The protocol takes approximately two hours for a 24 sample prep, with ~60 minutes of hands-on time, and requires 50 ng of starting DNA.
Because run output has zipped BCL files, you must use the bcl2fastq v1.8.4 conversion software to perform BCL to FASTQ conversion on your local Linux analysis system. This tool is run on Linux and has the same syntax, options, and functions (including demultiplexing) as the configureBclToFastq.pl script of CASAVA. The only difference is that it can be used to analyze either zipped or non-zipped BCL files.
If you send your data to BaseSpace, BCL to FASTQ conversion and demultiplexing are performed automatically following the completion of the data upload.
The ForenSeq DNA Signature Prep Kit contains two primer mixes to target specific regions of the genome. The mixes enable analysis of autosomal Y- and X-chromosome STR targets, identity-informative SNPs, with the option to include ancestry-informative, and phenotypic-informative SNPs. Libraries are prepared from an individual DNA sample, which can be multiplexed with libraries of other individual DNA libraries for sequencing on the MiSeq FGx Instrument.
Customer samples go through multiple quality control steps throughout the Illumina FastTrack Microarray Services workflow. The microarray team compares quality results to internal controls for accuracy verification. The microarray team takes these steps to ensure quality:
The Isaac aligner aligns reads by first identifying a small but complete set of relevant candidate mapping positions. The Isaac aligner begins with a seed-based search, using 32-mers as seeds. The initial single-seed search is followed by a multi-seed only for the reads that couldn't be placed unambiguously with a single seed. Speed up is achieved by sorting the reference index by the 32-mers. Improvement to accuracy is achieved by flagging of all the ambiguous reference positions in the index.
Following the seed-based search, selection of the best mapping among all the candidates is performed. For paired-end data sets, all mappings where only one end is aligned (orphan mappings) trigger a local search to find additional mapping candidates (shadow mappings) in the neighborhood defined by the expected minimum and maximum insert size. After optional trimming of low quality 3' ends and adaptor sequences, the possible mapping positions of each fragments are compared, taking into account pair-end information when available, possible gaps (using a banded Smith-Waterman gap aligner) and possible shadows. The selection is based on the Smith-Waterman score (using BWA, ELAND or user-defined scores) and on the log-probability of each mapping. The main speed-up comes from a parallel implementation of the gap aligner (using the SSE2 instruction set) and a shadow aligner optimized for short inserts. Further improvements could be achieved with AVX. The gapped alignment could be delegated to a coprocessor (e.g. Xeon Phi or GPU), however it is unclear if the benefit of large-scale parallelization would outweigh the cost of transferring the data between host and coprocessor.
Following alignment the fragments are sorted. Major speed-up in the sorting speed-up comes from efficient binning of the selected mappings, which greatly simplifies the sorting. Further analysis is performed to identify duplicates and optionally to re-align indels.
When both alleles of a heterozygous position are different from the reference, as in a tri-allelic position, the variants are split into two lines and both variants are annotated.
Cluster distance specifies the maximum distance that samples can be away from each other and still considered part of the same cluster. Increasing cluster distance will result in fewer clusters that are larger in size, while decreasing cluster distance will result in more clusters which are smaller in size. A cluster distance of 0.06 is typically a good starting point for initial clustering.
The Eco evaluation plate contains PCR primers that are designed to detect and quantify an artificial DNA sequence, with template DNA at defined quantities or no template at all. A standard curve with 20000, 10000, 5000, 2500, and 1250 copies in quadruplicate is used to quantify an unknown population of 24 replicates.
A user simply needs to add 20 µl of master mix to each well at a 1X concentration, incubate to fully resuspend the lyophilized primers and template, then centrifuge the plate and load it into the Eco. The Eco software comes with a preloaded template run file that includes the plate layout as well as the thermal cycling conditions.
Upon analysis, the data will show PCR efficiency, R2, standard deviation of replicates, and the melt curve analysis. The plate is intended for demonstration purposes, software training, and validation.
Temperature control and uniformity are the most challenging factors affecting HRM. The Tm shift for a single base change can be as small as 0.2° C for challenging Class IV A to T single nucleotide polymorphisms. Most current block-based instruments report temperature uniformity specifications in the range of ± 0.25° C to ± 0.5° C, generally considered too high to reliably differentiate a Class IV SNP. Block-based instruments that claim to perform HRM do so by extensive calibration and software compensation or by employing specialized analysis methods, such as temperature shifting, to overcome the thermal non-uniformity across the block.
The Eco system's unique thermal block provides thermal uniformity of ± 0.1° C, well above the industry standard for a block-based system. This extreme thermal uniformity allows the Eco system to perform HRM without software corrections. The thermal uniformity of the Eco system supports genotyping of even the most challenging Class IV SNPs.
Eco's hollow thermal block is constructed of highly conductive silver and is filled with a thermal conductive fluid. The fluid is rapidly circulated through the block by paired agitators that are powered by high-efficiency electromagnetic motors. The constant circulation of fluid evenly distributes the heat throughout the block to remove the hot or cold spots typical of block-based instruments. This design allows for rapid temperature ramps and extreme thermal uniformity (± 0.1° C) well to well across the plate, with no edge effect.
Customer samples go through multiple quality control steps throughout the Illumina FastTrack Sequencing Services workflow. The sequencing team compares quality results to internal controls for accuracy verification. The sequencing team performs quality checks at each of these steps:
Detailed results are provided in our white paper located here: http://res.illumina.com/documents/products/whitepapers/whitepaper_iassc_workflow.pdf
In summary: Isaac currently has slightly lower sensitivity and specificity, though it is much faster than BWA/GATK. Efforts to improve Isaac are ongoing and new versions will become available as methods progress.
Total number of SNP conflicts, SNP conflict rate, and sensitivity (% of non N reference sites called) of Isaac and BWA+GATK
TruSeq Stranded Total RNA Sample Prep Ribo-Zero kits remove ribosomal RNA (rRNA) using a hybridization/bead capture procedure that selectively binds rRNA species using biotinylated capture probes. The probe:rRNA hybrid is then captured by magnetic beads and removed using a magnet, leaving the desired rRNA-depleted RNA in solution.
One pair of oligos is designed for each amplicon. Hybridization of these oligos to unfragmented genomic DNA occurs in a 96‐well plate, followed by extension and ligation to form DNA templates consisting of the regions of interest flanked by universal primer sequences. Using indexed primers supplied with the kit, DNA templates are then PCR amplified, pooled into a single tube, and sequenced on the MiSeq System. For additional details on the assay, please refer to the User Guide.
Illumina does not have a specific schedule for updating the annotation database. This process largely depends on the cadence of updates from the different sources aggregated to create the annotation database.
Base calling and quality scoring are performed by a new implementation of Real-Time Analysis (RTA), called RTA 2, which includes important differences from RTA on other Illumina sequencing systems. For example, all processes are performed in memory to maximize processing speed, and configuration files and output file formats are different. For more information, see the NextSeq 500 System User Guide.
The workflow includes the following steps:
Cluster generation follows the same process that occurs on the cBot in preparation for runs on the HiSeq or Genome Analyzer, except on MiSeq all reagents for cluster generation, sequencing, and paired-end chemistry are loaded onto the instrument in a pre-filled reagent cartridge prior to starting the run. When the run is started, the MiSeq performs cluster generation followed by sequencing and paired end chemistry (if applicable).
Multiple samples can be pooled together because of the use of unique index (barcode) sequences added during library preparation. Indexes allow the software to separate out the data for each individual sample after sequencing.
Where CE-based assays give the length of the STRs, Illumina technology provides, not only the length of the STRs, but any variation in the STRs and genotypes at the various SNPs. Illumina technology also allows multiple samples to be pooled together and sequenced in one run, instead of running samples one at a time.
STR and SNP data are shown in multiple formats in the ForenSeq Universal Analysis Software. On the sample details screen, the genotype and/or warnings for each locus is displayed A detailed presentation is available for each locus by selecting it. For STR loci, a table of alleles is provided as well as a bar chart of the alleles and their relative intensities (# of reads). For SNP loci, a table of alleles is provided as well as a pie chart showing the distribution of the alleles.
The TruSeq ChIP Sample Prep kit generates Paired End (PE), indexed libraries that are compatible with all Illumina sequencing platforms and can be multiplexed. The legacy ChIP-Seq Sample Prep kit generates Single Read (SR) and non-indexed libraries that are compatible with Genome Analyzer, HiSeq and HiScanSQ, but not MiSeq.
The beta value (β) is used to estimate the methylation level of the CpG locus using the ratio of intensities between methylated and unmethylated alleles.
The first time the HCS 2.2 is launched, you will see a notification regarding instrument health data. This notification appears only once during the first initialization of the HCS, and will not appear again. Note that in pre-release, early access versions of HCS 2.0, this notification does not appear. However, instrument health agreement and notification is always available from Menu | Options | Tools, where you can also get more information and turn the option on or off.
The study number assigned to your samples is the next study number available in sequence.
Final pooled libraries can be stored at -25°C to -15°C for up to 3 months.
Prepare HiSeq v4 SBS reagents the night before or on the same day of use only. Do not store reagents longer than overnight. For use on the same day, store prepared reagents on ice in the original bottle with the cap tightened. For use the next day, store prepared reagents at 2°C to 8°C overnight.
You can store the rapid flow cell up to 24 hours after template hybridization and first extension. However, Illumina recommends that you start the sequencing run on the same day.
Maintenance wash solution can be stored for up to 30 days at room temperature. In that 30-day period, the solution can be used up to three times. For the first use, assign each bottle and tube to a reagent rack position. Maintain those positions for the second and third uses to prevent cross-contamination.
Prepare rapid SBS reagents the night before or on the same day of use only. Do not store reagents longer than overnight. For use on the same day, store prepared reagents on ice in the original bottle with the cap tightened. For use the next day, store prepared reagents at 2°C to 8°C overnight.
The post-run wash takes 30 minutes and a maintenance wash takes about 1 hour.
The LED arrays are designed to outlast the life of the instrument and are covered by the instrument warranty.
For a 2 x 250 bp run, analysis takes about 3 hours. This timing is dependent on running the latest PC RAM configuration on the MiSeq. This also depends on the genome size for resequencing. If analysis is taking longer than two hours, consider mapping to a more appropriate reference for your sample, or perform analysis offline by installing MiSeq Reporter on another computer.
If an alignment is performed against the whole genome, then the analysis time will be significantly longer than two hours. Also, bioinformatics analysis for metagenomics may take as long as 12 hours.
A 150-cycle run takes approximately 20 hours.
A 600-cycle run takes approximately 55 hours.
Generally, a run takes between 4 hours and approximately 55 hours depending on the number of cycles you perform. See the MiSeq System Product Information Sheet for complete information.
Run duration depends on the number of cycles sequenced, the type of flow cell, and whether the sequence is single or paired end. For example, a 2 x 150 bp run on a high-output flow cell takes approximately 29 hours, while a 1 x 75 bp run takes less than 11 hours. For more information, see the NextSeq 500 specifications page.
A water wash and maintenance wash each take approximately 1 hour. The maintenance wash protocol has changed to consist of only one step, washing the system with Tween 20 and ProClin 300, so there is no need to return to the instrument to reload wash solution or water.
It takes 1.5-2 days (approximately 30 hours) from genomic DNA input until libraries are ready to load on the flow cell, including approximately 5 hours of hands on time. Nextera Rapid Capture Enrichment libraries can be loaded on the flow cell the afternoon of day 2 of the protocol.
The post-run wash takes approximately 30 minutes.
Two washes are possible on the NextSeq using NCS v1.3: the automatic post-run wash and the instrument wash from the Welcome screen.
Preparing libraries takes approximately 9 hours. Sequencing and Real-Time Analysis (RTA) software tasks on the MiSeq FGx such as image processing, assigning base calls, and then designating a quality score for each base call takes approximately 30 hours. Aligning reads to provide sample information, along with optional steps such as generating reports, population statistics, sample comparison, phenotype estimations and bio-geographical ancestry estimations take approximately 1 hour.
It takes about 45 minutes to change from a high output (HiSeq v4 or TruSeq v3) to TruSeq Rapid mode. It takes about 3 hours to change from Rapid Run to a high output mode. Time spent for mode switching is in addition to instrument washing performed at the end of each run.
The sequencing run on the MiSeq FGx takes approximately 30 hours to complete, including RTA primary analysis.
Both Index 1 (i7) and Index 2 (i5) are each 8 bp in length.
The TruSight One 9-sample kit provides sufficient reagents to prepare a maximum of three (3) enrichment reactions of up-to 3-pooled samples per enrichment. The TruSight One 36-sample kits provide sufficient reagents to prepare a maximum of three (3) enrichment reactions of up-to 12-pooled samples per reaction. Single sample enrichments are possible, but the reagent volumes are optimized for preparing three sets of pooled sample libraries at either the 3- or 12-plex levels mentioned previously.
For dual index paired-end runs, there are 23 additional cycles (index & chemistry only).
For dual-index single-read runs, there are 16 additional cycles of indexing.
For information about the number of SBS kits required on the HiSeq, HiScanSQ, or GAIIx, see the user guide for your instrument guide.
The kit contains three boxes.
MiSeq v3 kits are kitted for 150 cycles and 600 cycles.
The NextSeq 500 can perform up to a 150-cycle paired-end run (2 x 150) using available NextSeq 500 kits. Kits are available in sizes of 300 cycles, 150 cycles, and 75 cycles, and each kit includes additional cycles for index reads. For more information, see the NextSeq 500 Kit Reference Guide.
HiSeq v4 SBS kits:
TruSeq v3 SBS kits:
HiSeq v4 SBS kits:
TruSeq v3 SBS kits:
HiSeq Rapid v2 SBS kits:
TruSeq Rapid SBS kits:
This depends on the aim of the experiment. For expression profiling projects, we do not recommend longer than 36 cycles of sequencing. The small RNA molecules are normally 15–30 bases long, and sequencing beyond this point only sequences the adapter. Eighteen cycles may be sufficient. For discovery projects, it may be worthwhile to do 40–50 cycles. This will sequence into the adapter, but the adapter sequence will be long enough to be unambiguously identified and removed even on longer small RNA molecules.
Index reads for single-read libraries use 7 cycle reads. Illumina does not support 6 cycle index reads for single-indexed libraries.
There are a total of 24 unique indices: 12 in Set A and 12 in Set B.
LT kits: 12 unique single indices in Set A and 12 unique single indices in Set B
HT kit: 96 unique dual indices
The NextSeq 500 flow cell contains four physical lanes. However, libraries are loaded onto the flow cell from a single reservoir. You can sequence one library or multiple pooled libraries on one flow cell.
The MiSeq flow cell is a single-lane flow cell.
Because each well of the 96-well HT adapter plate is single-use only, only one sample per index pair can be generated.
8–96 single-source database or reference samples can be processed and pooled and 8–32 casework or evidentiary samples can be processed and pooled.
This is dependent on the experiment plexity and MiSeq version. Illumina recommends using the TruSeq Targeted RNA Calculator to determine the number of samples that should be pooled together to achieve the depth of coverage required for your assay. Refer to the TruSeq Targeted RNA Expression tech note for information on how to design an experiment and determine the appropriate number of samples to be run on a flow cell.
TruSeq HT library prep kits support 96 samples. It is not recommended that the adapter plate undergo more than 4 freeze/thaw cycles. When less than the full set of 96 libraries are pooled and sequenced, it is important that libraries with compatible index combinations are used in the index pool. Please refer to the user guide for your TruSeq HT library prep kit for more information.
Each TruSeq ChIP Sample Prep kit contains enough reagents and adapters to process 48 samples. Set A and Set B each contain 12 unique indexes, with enough of each index sufficient for 8 individual samples.
The TruSeq DNA PCR-Free LT kits contain sufficient reagents for 24 samples and the TruSeq DNA PCR-Free HT kit contains reagents for 96 samples. Illumina recommends using the LT kit if processing less than 24 samples at a time and the HT kit if processing more than 24 samples. Both the LT kit and HT kit can be used with either the Low-Sample (LS) or High-Sample (HS) protocols.
The TruSeq Nano DNA LT Library Prep kits contain sufficient reagents for 24 samples and the TruSeq Nano DNA HT Library Prep Kit contains reagents for 96 samples. Illumina recommends using the LT kit if processing less than 24 samples at a time and the HT kit if processing more than 24 samples. Both the LT kit and HT kit can be used with either the Low-Sample (LS) or High-Sample (HS) protocols.
TruSeq DNA and RNA Sample Prep kits - Set A
TruSeq DNA and RNA Sample Prep kits - Set B
While Illumina supplies additional indexed adapters in the TruSeq Sample Prep v2 kits, the fill volumes are kept at 8 samples to minimize disruption to the protocols. The v2 kits are limited by other components to 48 samples per kit. This also offers more flexibility for how customers use the indexed adapters.
TruSeq Small RNA Library Prep kits are ordered as a core box and an index box. Each core box contains enough reagents to process 24 DNA samples and each index box contains 12 unique indexes, with sufficient index for two individual samples.
Scanning and analysis of a 2-lane rapid run flow cell creates 2 swaths per surface on 2 surfaces per lane. Each swath is divided into 16 tiles. For a 2-lane flow cell, there are a total of 128 tiles per flow cell.
Scanning and analysis of a high output flow cell is performed in 3 swaths per surface on 2 surfaces per lane. Each swath is divided into 16 tiles. Therefore, an 8-lane flow cell contains 768 tiles per flow cell.
There are two types of flow cells that can be sequenced on the NextSeq 500, the high-output flow cell and the mid-output flow cell. Both flow cells contain four lanes, but the lanes differ in width resulting in a different number of tiles.
For more information, see the NextSeq 500 System User Guide.
Going into the bisulfite conversion, at least 500 ng is required for the manual protocol, and at least 1000 ng is required for the automated protocol.
The ChIP-Seq Sample Preparation Kit is designed to use 10 ng of ChIP-enriched DNA. Many users have reported that using at least 30 ng of DNA makes the protocol simpler and more repeatable.
|Read Length||Estimated Rapid Run Time (Hrs)*|
|1 x 50 bp no index||9|
|1 x 50 bp dual index||11|
|2 x 100 bp no index||27|
|2 x 100 bp dual index||30|
|2 x 150 bp no index||40|
|2 x 50 1bp dual index||43|
|*Systems with SN < 7000895 will require additional time|
See system specifications on the HiSeq 2500 Specifications page.
Input Requirement Per Reaction
A maintenance wash is required every 10 days or when switching between high output and rapid modes. A water wash is required after each rapid run. After a high output run, you can choose between a water wash or a maintenance wash. Illumina recommends a maintenance wash.
We recommend quantitating ChIP DNA with the most sensitive method available, taking into account the low amount of DNA that is often available. Fluorescent dye-binding assays such as PicoGreen or Qubit offer high accuracy, assuming precise pipetting. The Bioanalyzer and High Sensitivity chips can also give good quantitation, and allow you to check the size and purity of your sample.
As of CASAVA v1.7, eland_rna uses the refFlat.txt.gz or seq_gene.md.gz file to generate the splice junction set automatically.
Illumina recommends that you check total RNA integrity following isolation using an Agilent Technologies 2100 Bioanalyzer with an RNA Integrity Number (RIN) value greater than or equal to eight.
Resuspension Buffer should be stored at -25ºC to -15ºC when first received. After the initial thaw, the reagent can be stored at 2ºC to 8ºC for use throughout the protocol.
The OncoArray consortium chose the selected SNPs specifically to develop a broad-use array platform that provides a detailed querying of:
Check http://www.illumina.com/ecoqpcr regularly to find out about new Eco system developments and applications. In addition, read the Illumina monthly customer newsletter, Illuminotes, to be promptly notified of any changes to systems, applications, or documentation.
You can filter by Index in SAV (Sequencing Analysis Viewer) for indexed runs.
If dual-index libraries are combined with single-index libraries on the same flow cell, single-indexed libraries will have sequence that can be ignored for the second index read. For this combination, you need to create two sample sheets: one sample sheet for lanes containing single-index libraries and another sample sheet for lanes containing dual-index libraries. Run CASAVA separately with the appropriate --use-bases-mask command for the index reads to demultiplex appropriately. See Sequencing Mixed Libraries on a HiSeq or GA Flow Cell.
Yes. The new MiSeq software package is backward compatible with v2 reagents. Using the RFID feature, the MiSeq automatically recognizes which kit version is loaded for the run and chooses the appropriate Q-table. There are no changes to v2 workflows.
In some cases it is possible to convert existing traditional PCR assays into Real-Time PCR assays, with a few considerations around primer design and master mix. Primer design is one of the first considerations for converting a traditional PCR assay. Real-Time PCR is most efficient with relatively short amplicon lengths, in the range of 50 to 150 bp. Longer products can be used if the cycling conditions are changed to accommodate longer extension times, but you should avoid products longer than 300 bp. In some cases it might be possible to design a TaqMan probe to hybridize between the two existing PCR primers. If not, you can use SYBR Green I for detection. (See FAQs What are the advantages and disadvantages of TaqMan and SYBR Green I chemistries? and Are SYBR Green I Real-Time PCR assays less specific than TaqMan probe assays? for more information on TaqMan and SYBR Green I.)
The master mix is another consideration when converting a traditional PCR assay into a Real-Time PCR assay. If a TaqMan probe can be designed, you might be able to use the same master mix that was used for the traditional PCR assay. If a TaqMan probe cannot be designed, you should add SYBR Green I to the master mix. In either case, a certain amount of optimization may be needed to obtain good Real-Time PCR results.
Illumina has discontined the TruSeq DNA Sample Prep kits. The Sample Preparation: Kit Selector can assist in choosing whether the TruSeq DNA PCR-Free Library Prep or TruSeq Nano DNA Library Prep kit best fits your needs. In summary, the kit selected depends on available sample input amounts and the quality of data.
The Eco sample loading dock provides a backlight that highlights the content of the wells. The light increases the contrast of the alpha numeric sample locators and helps you monitor sample loading. The dock has an adjustable foot that lets you angle the plate for optimal visibility if needed; for example, when loading under a laminar flow hood.
On the Plate Layout tab, click the Assays button to open the Assays dialog box. Add two assays, one for the target and another for the IPC. Assign reporter dyes from different channels to each assay.
Regardless of the plate setup, the Eco system acquires data in all four channels for all 48 wells. The dyes enable you to focus on the desired information during analysis.
Data can be exported directly from the Samples Table, SNP Table, and Full Data Table for downstream analysis. Mark the columns and rows you wish to export and click the icon for "Export displayed data to file" to save selected table content in *.txt or *.csv format.
See Chapter 5 of the GenomeStudio 2008.1 Framework User Guide, available on iCom and in the GenomeStudio Portal.
Yes, please download the GenomeStudio 2011.1 installer from the Illumina website. It is sufficient to install the GenomeStudio Framework by clicking the respective box in the install wizard, which does not require a license key. It is not required to install the GenomeStudio Genotyping Module on the same computer on which the PC Module is installed. However, the polyploid workflow does require generating a genotyping project in the GenomeStudio Genotyping Module prior to taking the data to the PC module for polyploidy clustering.
If you run into the ERROR: reference sequence ⁄data⁄runs⁄genomes_human⁄c1.fa does not exist, there is a good chance that you are not using files provided by Illumina.
Assuming that you have specified the reference genome to be: --refSequences=/data/runs/genomes_human and genome size file: -g conf/human_rna_size.xml you have three options:
1. Use the human reference genome provided by Illumina. This is preferable since the GenomeStudio Software assumes human reference genome provided by Illumina.
2. Change the names of your fasta files in ⁄data⁄runs⁄genomes_human to match the names in human_rna_size.xml.
3. Instead of using conf⁄human_rna_size.xml, use genome_sizes.xml produced by Pipeline.
NOTE: For CASAVA for RNA Sequencing all reference sequences and genome_size files are provided by Illumina.
The warning message "ARM9BoardSerialPort (ARM9CHEM): timed out waiting" indicates that an ARM9 communication time out has occurred. The ARM9 board is one of many components that communicate between the HiSeq and instrument computer. Messages related to an ARM9 time out are not necessarily indicative of a hardware issue, and do not impact the run or data quality.
If this message appears repeatedly, perform a normal stop on the current run, shut down the HCS/RTA software, and then power cycle the HiSeq and instrument computer to reestablish communication between the systems. Launch HCS and resume your run. Continue to monitor your run to make sure that the issue is resolved. If it appears that the run data is affected, contact Illumina Technical Support for further assistance.
TDI Scan warning messages indicate an issue with image acquisition and storage; however, the system will automatically retry image capture to self-correct. TdiScan messages usually have no effect on the run other than slightly extended cycle times, and do not affect the run data as images are re-captured before continuing.
In the rare event that the retry threshold is exceeded, one imaging swath is skipped for one cycle. If this message occurs frequently, contact Illumina Technical Support for assistance.
All uploaded data is initially deposited in a staging area until it is reviewed by Illumina personnel. Data is not released to iControlDB until it has undergone this review process, which may take up to 10 business days. Additionally, data will not be released if they do not pass our call rate threshold of 98%. If you still do not see your data after 10 business days, please contact email@example.com for further investigation.
This amount of free space is required at the beginning of a run. The system assumes that data are transferred to the network copy of the run folder in real time. Therefore, 750 GB is the safe level to start a run. The software assumes that the run copies and deletes the files as they are processed, and that the connection to the network server can keep up with file transfer.
Although the Illumina adapters are designed to reduce adapter-adapter ligation, very low amounts of starting material can result in high adapter-insert ratios during ligation. This promotes formation of adapter dimers and occasional adapter concatamers. These concatamers often take the form of amplified trimers in various configurations. They can be removed by careful size selection, or by repeating the ligation with more input DNA or less adapter. The amount of adapter added to the ligation can be titrated by additional dilution (i.e. a 20× or 50× dilution, rather than the 10× dilution described in the protocol).
Coverage of GC regions can be impacted by the model, settings, and performance of the thermal cycler used. Illumina has validated the Bio-Rad DNA Engine Tetrad 2, the Bio-Rad S1000, and the MJ Research PTC-225 DNA Engine Tetrad. Other thermal cyclers may differ in their performance across the genome.
SNPs are only clustered for samples selected in the Samples Table (marked in blue). If no samples are selected in the Samples Table, SNPs are clustered for all samples in the Samples Table (except non-excluded samples). Thus, if you wish to cluster SNPs for all your non-excluded samples please make sure that no samples are selected in the Samples Table at the time of clustering (e.g. by clicking onto an area in the SNP graph).
We review the data for many criteria before releasing to the database. Any samples that do not pass our call rate criteria are not included in the released data. Additionally, there may be user-entered information (e.g., ethnicity or positive phenotype) that is unclear or not compliant with the Health Insurance Portability and Accountability Act (HIPAA). These samples will not be included in the released data.
TruSeq kits support many low plex pooling options across the entire plate. Some of these combinations are outlined in the Pooling Guidelines section of the appropriate TruSeq library prep guide. Customers can also design their own color-balanced pools, but we highly recommend that the Illumina Experiment Manager be used to check the color balance of user-designed pools
You can upgrade directly to HCS1.5/RTA1.13 from HCS1.4/RTA1.12. If you are running an older version of HCS, please contact Illumina Technical Support for assistance in upgrading.
No. VariantStudio v2.1 is programmed to use annotations from the previous version of the Illumina Annotation Service. It does not connect to the new version that is available with VariantStudio v2.2. If you want to use the new version of the Illumina Annotation Service, use VariantStudio v2.2 and reannotate any samples in projects from VariantStudio v2.1.
If I have custom content, can I get help with SNP selection? The project manager helps you design your SNP panel. In this collaborative process, the project manager uses Illumina design software to assess feasibility of SNP lists that you provide in the form of rsIDs or sequence submissions for novel SNPs or non-human panels. For each submission, the project manager provides a feasibility assessment as well as tag marker selection for haplotype blocks and gene annotations. After you select your final SNP list, the project manager takes the responsibility to order the beadpool for you.
You can import external sources of annotations into VariantStudio using the custom annotation feature. Check your HGMD Professional or HGMD public version license terms to make sure that such use is permitted.
No. If samples are reannotated in VariantStudio v2.2, all annotations for variants are replaced with the annotations from the newer version of the Illumina Annotation Service. If you do not want to lose previous annotations for these samples, create a project for the samples that you want to preserve with the old annotations.
No. You can start each flow cell independently from the other. Each flow cell can have a different number of reads and cycles.
No. VariantStudio v2.2 is programmed to use annotations from the version of the Illumina Annotation Service that was updated for VariantStudio v2.2.
Yes. HCS 2.2 is available for all customers.
MiSeq offers scalable throughput based on read length. Illumina continues to increase read lengths, imaging area, and cluster density with improved detection and resolution. For example, at launch the MiSeq performed up to 150-cycle paired-end runs (2 x 150 bp) with greater than five million reads passing filter. Currently, the MiSeq can perform up to 250-cycle paired-end runs (2 x 250 bp) that generates 15 million reads passing filter. For more information, see the MiSeq Product Information Sheet.
An instrument wash is performed automatically when the run completes. For this wash, a dilute solution of 0.03%-0.06% NaOCl is required. To prepare for the automatic post-run wash, load 3 ml NaOCl in reservoir #28 before loading the reagent cartridge.
A manual wash is performed when required from the Welcome screen. As of the release of NextSeq Control Software v1.2, or later, the addition of NaOCl is no longer required for the manual wash. If you are using an earlier version of the control software, the software expects 3 ml 0.03%-0.06% NaOCl in position #28 of the reagent wash tray.
The Directional mRNA-Seq Sample Preparation Guide provides instructions to use the Illumina Small RNA adapters, which are specific to the 3' and 5' fragment end, to generate a stranded library with the original Illumina mRNA-Seq kit. The protocol has not been optimized for the TruSeq RNA Sample Prep Kit and Illumina does not recommend this use of the TruSeq RNA Sample Prep Kit.
hg19 + refseqs transcript model
dbSNP build 137, common SNPs ≥ 1% MAF
mm9, mm10 + refseq transcript model
dbSNP build 128 for mm9, dbSNP build 137 (common SNPs) for mm10
dbSNP build 125 for rn4
No. Illumina VariantStudio was is a point-and-click software application that enables variant data exploration, annotation, and filtering without requiring bioinformatics expertise.
If you are using the NextSeq 500 in standalone mode, use IEM version 1.8.2 to create a sample sheet for the NextSeq.
If you are connected to BaseSpace, use the BaseSpace Prep tab to record library and indexing information, and specify other run parameters. When runs are set up on the Prep tab, the run name appears as a selection during the run setup steps on the instrument.
No, the reagent blank is made by the kit user.
No. The NextSeq does not expect a sample sheet during run setup. When connected to BaseSpace, library and indexing information is entered on the BaseSpace Prep tab before the run, and the information is passed to the NextSeq. The available run names appear on the instrument screen during the run setup steps.
However, if you are using the NextSeq 500 in standalone mode (not connected to BaseSpace), use Illumina Experiment Manager (IEM) v1.8.2, or later, to create a sample sheet for the NextSeq.
For runs on the HiSeq, HiScanSQ, or GAIIx, creating and loading a sample sheet at the start of the run is optional. However, using a sample sheet allows you to view data shown on the indexing tab in the Sequencing Analysis Viewer (SAV) during the run. If you do not load a sample sheet at the start of a run in HCS, you will not be able to view indexing data in SAV. When analyzing indexed samples using CASAVA v1.8.2, a sample sheet is required. MiSeq runs require a sample sheet when setting up the run in MCS.
Illumina recommends that you create the sample sheet using the Illumina Experiment Manager (IEM) prior to performing library prep in order to confirm appropriate index combinations.
Contamination is always a concern with PCR. Illumina recommends setting up PCR1 in a Pre-PCR facility. Illumina recommends using two areas in the post-PCR area: one for setting up PCR2 (no libraries present in this area) and a separate area for the library purification, bead-based normalization, and MiSeq FGx set-up.
Only LT adapters are methylated and the polymerases used in the TruSeq kits are not appropriate for bisulfite applications.
If there is no signal in one of the color channels of the index read, the image registration might fail and no base will be called from that cycle. If no base is called, the index read may not be able to be matched to the sequence specified in the sample sheet, and then samples will not be able to be demultiplexed.
Currently this is not supported by the Eco system. Check back periodically and keep an eye on the monthly Illuminotes newsletter for updates.
The EcoStudy software, which is available for Eco Real-Time PCR System users, is able to apply a standard curve from one plate across an entire study for data analysis. The software is also able to import an existing standard curve from a previous study for use in analysis.
Samples are listed as being submitted either by Illumina, or by Other. The User Agreement that is necessary to comply with the Health Insurance Portability and Accountability Act (HIPAA) does not allow the identification of the source of the data from the download tool. However, it is projected that many submitters will reference their study number in their publications.
The initial PCR amplification for Quantitative Fluorescence PCR (QF-PCR) can be performed on the Eco system. The subsequent detection and quantification of the amplified fragments requires electrophoresis on a DNA scanner. Some applications of QF-PCR, such as Copy Number Variation (CNV) analysis, can be adapted into Real-Time PCR and be performed on the Eco system.
Illumina has tested only RNA isolated using RNEasy® from Qiagen®, one of the most frequently used methods. We do not anticipate a major impact on performance with other appropriate, well-established isolation techniques.
Samples can be quantified prior to pooling, but this process is often inaccurate. At high plexity, it may be more effective to pool using equal small volumes prior to gel purification then purify, amplify and quantitate the pooled library. Once the pool is sequenced, any samples that did not give sufficient coverage can be re-pooled and sequenced.
Quantification is performed before pooling the libraries and must be done using qPCR. Other methods will quantify fragments that may not have adapters at both ends and will thus not generate clusters or sequence. Quantification by methods other than qPCR will be inaccurate. It is possible to quantify after pooling if all DNA samples are of similar quality, but this requires very consistent yields and should not be attempted by a new user. See the TruSeq DNA PCR-Free Library Prep Guide for details.
Currently, the Eco system is only available in a 48-well format.
One complete sample loading dock is provided with each Eco system.
To purchase additional docks:
2. Click the Options & Accessories tab
3. Scroll down to the Eco sample loading dock field
4. Place your order
No. CASAVA 1.8 generates BAM files which are not compatible with the current version of the GenomeStudio ChIP-Seq Module. Investigators using the ChIP-Seq Module should run CASAVA 1.7.
This is a two-color assay.
For the Infinium HumanMethyation27 BeadChip assay, which is based on Infinium I Assay designs, the color incorporated depends upon the base preceding the CpG locus being queried. This can be either green or red.
The Infinium HumanMethylation450 BeadChip assay includes Infinium I and Infinium II study designs. In the latter case, a single base extension from the 3' end of the probe sequence (which is one base upstream of the query base) will result in either a red or green signal depending on whether the query site was unmethylated or methylated.
The sequencing team runs each of your samples in parallel on the HumanOmni2.5 genotyping BeadChip. You receive a file of the genotyped SNPs in VCF format. Illumina GenomeStudio software directly outputs the VCF file. The calls are obtained by applying the product standard cluster file to your data. For projects larger than 100 samples, Illumina recommends that you recluster on the project samples, check SNP performance, and re-export the genotype calls.
Illumina also provides intensity data files (IDATs) and a sample sheet. You can combine these files with the product definition files (beadpool manifest .bpm, and standard cluster file .egt) that are found on the Illumina support website to recreate a GenomeStudio project from the source data. This step allows you to visualize and assess SNP and sample performance.
Yes, the TruSeq DNA PCR-Free Library Prep kits will give better coverage for difficult to sequence regions. Please see the TruSeq DNA PCR-Free Library Prep Kit Datasheet and example datasets for more detailed information.
The TruSeq Nano DNA kit is not compatible with the TruSeq Exome Enrichment or Custom Enrichment kits. The Nextera Rapid Capture product supports a variety of enrichment applications. For more information, see Nextera Rapid Capture.
Yes, BaseSpace can be used for the analysis of TruSight One runs. Illumina recommends using the Illumina Experiment Manager and select Targeted Sequencing and the subsequent Enrichment workflow. Download VCF files from BaseSpace projects to filter variants using the VariantStudio software.
No. The VariantStudio software was developed as a Research Use Only tool.
Illumina VariantStudio has undergone standard software development and testing requirements applied to all Illumina software tools developed for Research Use Only.
Illumina has not submitted the Illumina VariantStudio software for regulatory approval. The software has not undergone analytical validation or clinical validation required for software tools that are regulated as a medical device. Therefore, it is important that you follow procedures for validation of the software according to your Institution, Local, State, and Federal guidelines.
Illumina recommends performing the gel size selection step as it is designed to remove unligated adapters, as well as any adapters that might have ligated to one another, and selects a narrow 250–300 bp size-range of DNA fragments for ChIP library construction appropriate for cluster generation.
Yes. The incorporation reagent included in the HiSeq v4 SBS kit appears bluer, instead of the purple color of incorporation reagents included in the TruSeq v3 SBS kit.
No. The HiScanSQ runs HCS 1.5.15 and is not upgradeable.
If you are using CASAVA, it is compatible. However, bcl2fastq v1.8.4 must be used in place of the configureBcl2fastq step in CASAVA. The output of bcl2fastq v1.8.4 is in the fastq.gz file format organized into project and sample directories as specified in the sample sheet. This output is compatible with the configureAlignment and configureBuild components of CASAVA v1.8.2. The sample sheet format required for bcl2fastq v1.8.4 is equivalent to CASAVA v1.8.2 sample sheet format, and is described in the bcl2fastq v1.8.4 User Guide (part # 15038058).
If you are not using CASAVA, note that Illumina is discontinuing distribution of CASAVA software to better support new products available on BaseSpace. BaseSpace features analysis options for a large array of NGS applications.
LIMS support is available for the Infinium HumanMethyation450 BeadChip.
No. Illumina recommends purchasing a 600-cycle kit and discarding the excess reagents after the run. Because of the increased cluster density enabled by v3 runs, this is more economical than running fewer samples with MiSeq v2 reagents.
Since Infinium Methylation arrays are designed to compare relative methylation levels between two samples or sample groups (such as normal versus tumor, or pancreas cells versus liver cells), there is no GenTrain and/or cluster file for this product. It is similar to doing a paired-sample analysis.
The new index adapter design enables PCR-free protocols. (A single cycle of synthesis is required to separate the forked adapter.) However, for applications that require higher amounts of input for sequencing, Illumina recommends 10 cycles of PCR (with an optional titration for potential cycle reduction and to optimize throughput). The TruSeq DNA LT/HT kits (FC-121-2001, FC-121-2002, FC-121-2003) use 10 cycles of PCR. The TruSeq DNA PCR-Free LT/HT kits (FC-121-3001, FC-121-3002, FC-121-3003) are optimized for PCR-free applications. These kits and protocols are not interchangeable. Please see the TruSeq DNA PCR-Free HT Sample Prep Kit support pages and the TruSeq DNA PCR-Free LT Sample Prep Kit support pages for more information.
No, the pricing remains the same.
Yes, in the SNP graph, use the curser to draw a box around the samples you wish to manually edit, right-click and choose the cluster samples should be assigned to, or NC (no call) if you wish to remove samples from any clusters.
Processing fewer than 8 samples at one time, including positive and negative controls, can cause problems with pipetting accuracy, due to the small volumes used when preparing the master mix.
An applicator tool (squeegee) is included with each Eco sample loading dock.
Projects that are in-progress may keep their current configuration and workflow rather than changing protocols in the middle of the study. Additionally, pooling reads from paired-end stranded runs is equivalent to the same number of reads from non-stranded single read runs (i.e., 2 million reads each of read 1 and read 2 pooled together (4M total) is identical to 4 million reads of read 1 of a non-directional library).
Introducing too much DNA onto the flow cell results in a raw cluster density over the recommended 1.4M/mm2 threshold. Overclustered flow cells can result in poor quality data, as individual clusters cannot be adequately resolved and low numbers of clusters pass filters to be used in final output. It is recommended to target < 1.4M/mm2 to ensure high-quality sequencing performance.
Sequencing 550 bp inserts is not currently compatible with the NextSeq 500.
Yes. OPAs are shipped at ambient temperature; stability testing indicates that OPAs are quite stable for short term storage (3-4 weeks) under these conditions. For long-term storage (> 1 month), we recommend storage at -20ºC.
There are three options:
--Download GenomeStudio software from iCom.
--Install GenomeStudio software over a network that has a shared DVD drive or a copy of the GenomeStudio image.
--Purchase a portable DVD drive with a USB port, then install GenomeStudio software from the DVD.
The Eco optical system components do not move during operation, with the exception of the filter slide, which moves while measuring the four emission wavelengths for each sample at each cycle.
The optical system is calibrated prior to shipment and does not require recalibration. An optional yearly calibration plan is available. If your application requires annual validation, contact Customer Service at 1.800.809.4566 in the U.S. or email firstname.lastname@example.org for pricing and assistance.
The Eco system comes with five licenses for the system software, so that you are not constrained to perform the data analysis on the included workstation. You can install the Eco software from the USB drive that is provided with each Eco. If you require more licenses, contact Illumina customer service.
Illumina recommends quantitation of amplified RNA by fluorometry using Molecular Probe's RiboGreen® Reagent. We also recommend additional qualitative analysis with the Agilent Bioanalyzer or by electrophoresis through agarose gel. A less precise RNA quantitation method is the measurement of A260 absorbance with a spectrophotometer.
RNA libraries should routinely be run as single read (with an index read as desired). TruSeq libraries can be run as paired-end if desired (for example, for directional RNA custom preparations), but this is expected to offer no advantage and significant expense with small RNA libraries.
The Eco system's maximum power draw is 500 VA, which determines the VA rating of the UPS: > 500 VA.
The average power draw during aggressive Eco cycling is less than 300 VA, which determines the UPS capacity requirement.
If you want to be able to continue a run during a power failure, then the APC UPS (Part # SUA2200 or SMT2200) is a suitable model, providing approximately 3 hours at 10% of max capacity.
Illumina has not tested kits designed to label microbial RNA. Reagent vendors such as Ambion® do sell kits for this application. These kits may work, but there is no particular kit that we recommend. It is important that a single source of biotin-16-UTP (i.e., same vendor) is used for all labeling reactions (e.g., Ambion #8452 or #8453).
The Eco optical system supports dyes within four channels, ranging from 505 to 705 nm. The optical system can detect any dyes that fall within those ranges.
Example Fluorophores Detected
SYBR Green I, FAM
Illumina recommends using Coriell Human-1 DNA (NA18507) or Promega Human Genomic DNA (G3041) as a positive control for this protocol.
During a typical run, Real Time Analysis (RTA) may occasionally run into copy issues, and will retry copying the file later. When the file is successfully copied, RTA proceeds normally. Therefore, seeing a few of these issues in the error log is acceptable. However, if you see many of these entries (dozens or hundreds), there could be a problem with your network. You might have an incorrect password , network path, or your network connection might not be fast enough. Try the following to troubleshoot this issue:
When you downloaded iControlDB, you received template files that can be used in place of a Final Case Report. These template files do not contain sample genotypes, but are in the format required by iControlDB.
A list of the Illumina barcodes and their corresponding HapMap IDs is available upon request from email@example.com.
This is the HumanHap550v1 product.
For genotyping applications, Illumina recommends use of PicoGreen reagent for DNA quantitation. UV-based methods may overestimate DNA concentration by 2–10 fold.
TruSeq ChIP libraries are compatible with all Illumina sequencing platforms, including MiSeq, HiSeq, HiScanSQ and Genome Analyzer.
CASAVA and (MSR) can be used for demultiplexing. However, they are not intended for TruSeq ChIP analysis.
Nextera Rapid Capture Exome Enrichment (8 rxn × 1 plex)
Nextera Rapid Capture Exome Enrichment (8 rxn × 3 plex)
Nextera Rapid Capture Exome Enrichment (8 rxn × 6 plex)
Nextera Rapid Capture Exome Enrichment (8 rxn × 9 plex)
Nextera Rapid Capture Exome Enrichment (2 rxn × 12 plex)
Nextera Rapid Capture Exome Enrichment (4 rxn × 12 plex)
Nextera Rapid Capture Exome Enrichment (8 rxn × 12 plex)
Nextera Rapid Capture Expanded Exome Enrichment (2 rxn × 12 plex)
Nextera Rapid Capture Expanded Exome Enrichment (4 rxn × 12 plex)
Nextera Rapid Capture Expanded Exome Enrichment (8 rxn × 12 plex)
Nextera Rapid Capture Custom Enrichment (48 Samples)
Nextera Rapid Capture Custom Enrichment (96 Samples)
Nextera Rapid Capture Custom Enrichment (288 Samples)
For STR typing, ForenSeq Universal Analysis Software presents a locus QC flag when more alleles are present than expected, the alleles are not balanced, an allele is below the interpretation threshold but above the analytical threshold, the number of reads was not enough to make a call, or when stutter is present above the allowable level for the locus. For SNP typing, ForenSeq Universal Analysis Software presents a locus QC flag when the alleles are not balanced, an allele is below the interpretation threshold, but above the analytical threshold, or when the number of reads was not enough to make a call.
TruSight One Sequencing Panel (9 samples). Includes MiSeq reagents.
TruSight One Sequencing Panel (36 samples). Sample Prep only.
TG TruSight One Sequencing Panel (9 samples). Includes MiSeq reagents.
TG TruSight One Sequencing Panel (36 samples). Sample prep only.
The MiSeq includes all the hardware needed for cluster generation, sequencing, and data analysis. More advanced analysis requires additional computing infrastructure. Other equipment may vary with application and sample prep methods, which is outlined in sample prep documentation.
The HiSeq X system is intended for large-scale human whole-genome sequencing projects using either the TruSeq Nano DNA (HT or LT) or the TruSeq DNA PCR-Free (HT or LT) library prep kits. Other applications and species are not compatible or supported on the HiSeq X system.
The MiSeq system is ideal for amplicon sequencing, targeted resequencing, small genome sequencing, and clone checking. It is capable of performing 16S ribosomal RNA gene sequencing, ChIP-Seq (TF Binding), and small RNA sequencing.
The ForenSeq DNA Signature Prep Kit is used for two primary applications: Criminal Cases and Database, but can also be used in other applications, such as Missing Persons, Mass Fatality, and Bio-geographical Ancestry.
The MiSeq supports a large portfolio of sequencing applications. See the MiSeq Applications page for more information.
Illumina FastTrack Services combine high-performance Illumina platforms with expert Illumina scientists. Illumina FastTrack Services deliver high-quality genotyping and sequencing data to support your research projects. You reap the benefits of Illumina technology with a personalized service that delivers your data quickly and at a reasonable cost.
Illumina FastTrack Services offer a broad range of services, from whole-genome genotyping to custom content genotyping, human whole-genome sequencing, and human phasing analysis services.
We do not have any specific recommendations regarding the chromatin immunoprecipitation step, as techniques for this process can vary widely based on the desired application. However, we do not recommend using any type of nucleic acid as a carrier in the chromatin immunoprecipitation prior to sequencing. The carrier nucleic acid is difficult to remove from the sample, and can end up being 50–90% of the final library.
The table below includes Illumina’s minimum hardware recommendations to run GenomeStudio software.
|Parameter||Sequencing Data Analysis||Microarray Data Analysis||Microarray and Sequencing Data Analysis|
|CPU Speed||Intel Pentium 2.0 GHz or faster||Intel Celeron Duo or faster||Intel Celeron Duo or faster|
|Memory Size||4 GB or more||8 GB or more||8 GB or more|
|Hard Drive||250 GB or larger||100 GB or larger||250 GB or larger|
|Video Display||1,280 x 1,024||1,280 x 1,024||1,280 x 1,024|
|Operating System||Windows XP, Vista, or Windows 7||Windows XP, Vista, or Windows 7||Windows XP, Vista, or Windows 7
|Specific OS Requirements||Microsoft .NET Framework 3.5||Microsoft .NET Framework 3.5||Microsoft .NET Framework 3.5|
|Network Connection||1 GbE or faster||1 GbE or faster||1 GbE or faster|
Accurate library quantification is needed to achieve even pooling for enrichment as well as the recommended raw cluster densities. Inaccurate quantitation and pooling can result in higher representation of some samples compared to others in the same pool. Accurate quantification of the final library before loading is needed to reach the recommended raw cluster densities. Illumina recommends using the same diluted library for both quantitation as well as clustering. If cluster densities are lower than expected, Illumina recommends checking the library size used in the concentration calculation using a Bioanalyzer trace and adjust accordingly.
There are six safe stopping points in the Nextera Rapid Capture Enrichment protocol:
There are eight safe stopping points in the TruSeq RNA Access Library Prep protocol:
Some basic Real-Time PCR terms and their definitions are:
Amplification plot—Plot of fluorescent signal versus cycle number.
Baseline—The initial cycles of PCR where there is little to no change in fluorescence.
Threshold—The arbitrary level of fluorescence used for Cq determination. Should be set above the baseline and within the exponential growth phase of the amplification plot.
Cq (quantification cycle)—The fractional cycle number where fluorescence increases above the threshold. Also referred to as Ct (threshold cycle) or Cp (quantification cycle).
Rn—Normalized reporter signal.
ΔRn—Baseline subtracted normalized reporter signal.
Slope—Indicates the efficiency of the reaction. With 10-fold dilutions, a slope of -3.32 indicates a perfect doubling of product per cycle (100% PCR efficiency).
R2—Reports the linearity of the standard curve.
With Infinium products, the two main parameters for copy number are the B Allele Frequency (based on genotypes) and the Log R Ratio (based on intensities). The Log R Ratio is the log (base two) of the "observed intensity" divided by the "expected intensity". The "expected" intensity is generated from the cluster file.
Because of this direct comparison, accurately measuring the sample input amount is vital. Essentially, your input amount should match the recommended value (400ng for Infinium HD Duo; 200ng for Infinium HD Quad and Infinium HD 12-sample products). If this is this case, your Log R Ratio signal will tend to have low noise.When the DNA samples are inaccurately quantified, you may see an "undulation" pattern (this looks like waves) in the log R ratio. This tends to be in GC-rich regions of the genome. This wavy pattern makes it difficult to do CNV analysis as the waves themselves look like copy number changes. This tends to confuse algorithms and confound analysis. On the other hand, call rates tend to be only slightly affected by this change (but this varies).
There are a number of third party solutions available for the analysis of ChIP data. These include, but are not limited to, MACS, Avadis, and Partek. Please note that Illumina cannot provide support for the use of third party software; please contact the software resources directly with any questions regarding the analysis and use of their software. Additional literature references can be found on the Illumina Epigenetics webpage.
Illumina recommends 50 μl input DNA at 10 ng/μl.
For more information, see DNA Input Recommendations in the TruSeq Synthetic Long-Read DNA Library Prep Guide.
Follow these DNA input recommendations:
The Eco system runs from a standard wall socket over a wide voltage range (100–240VAC). It does not require 3-phase power. The Eco system ships with a European line cord and a U.S. line cord. The local distributor should provide an appropriate line cord for local power outlet configuration.
Temperature Operating Range 15° to 30° C (59°F to 86° F)
Storage 10° to 38°C (50°F to 100°F)
Humidity Operating Range 15–90% RHStorage 5–95% RH
Illumina provides each customer with a seat license for Illumina GenomeStudio software. Illumina provides all samples and markers in a GenomeStudio project workspace. The Project Scientist zeros poorly performing samples and markers. These samples and markers remain available, enabling customers to make their own determination. Illumina provides genotyping data files, which indicate the bi-allelic genotyping call with each genotype on a separate row. The exported data files are customizable, and the Project Scientist asks customers for their preference before delivery. Optional data fields include intensity values, allelic strand formats, etc. The deliverables also include intensity files and locus and DNA summary files that show project statistics. Illumina delivers all data via a secure FTP site. The project scientist provides ongoing support for questions about the data.
The WGS analysis pipeline v3.0 uses Isaac Aligner, and Isaac Variant Caller to generate several outputs. These outputs include sequencing reads with reduced-resolution Q-scores in BAM format, and variant data in both VCF and genome VCF (gVCF)1 file format. The somatic small-variant calling component of the cancer analysis pipeline uses Isaac Aligner and Strelka2 to generate somatic small-variant data in VCF format. These informatics pipelines enable significantly increased alignment efficiencies and reduced data footprints, without compromising the quality of the data and variant calls.
A WGS sequencing analysis training package, a cancer analysis training package, and a human phasing training package are available in BaseSpace. A services user guideline and a quick video describing the deliverables are also available in BaseSpace. See the Sequencing Services Training Material for more information.
The main advantage of TaqMan chemistry is that a fluorescent signal is generated only when there is specific hybridization of the probe to the target sequence. No signal is generated from any non-specific amplification products that were formed during the reaction. Another advantage is that probes can be labeled with different, spectrally distinct reporter dyes, which allows the amplification of multiple target sequences within a single tube (multiplex Real-Time PCR). The main disadvantage of TaqMan chemistry is that design and synthesis of different dual-labeled probes is required for each target sequence, which increases assay setup and cost.
The main advantage of SYBR Green I chemistry is that it only requires the design and synthesis of two PCR primers, which decreases assay setup and cost. Another advantage for SYBR Green I chemistry is the ability to perform melt curves. The main disadvantage of SYBR Green I chemistry is that since SYBR Green I binds to any dsDNA present during the reaction it will bind to and generate a signal for any non-specific amplification that occurs.
One advantage of Real-Time PCR over traditional PCR is that it is a closed-tube system requiring no post-PCR processing. Real-Time PCR has higher precision, increased sensitivity (down to one copy), increased dynamic range (greater than 8 logs), and high resolution (less than two-fold differences).
The Nextera Mate Pair Gel-Plus protocol with size-selection aims to generate mate pair libraries with a narrower size distribution and larger median fragment size. This is achieved by using a gel-based size-selection process to select DNA of a desired size range. A Mate Pair library with a narrow distribution of fragments will facilitate accurate structural variation detection. It will also help de novo assemblies of both simple and complex genomes by providing paired read information which spans larger repeat regions.
However, the degree of difficulty in generating gel-plus libraries increases as the length of fragments increases. Compared to libraries with smaller Mate Pair fragment sizes, libraries with larger fragment lengths are expected to have a lower final library yield and lower library diversity and are more challenging to make robustly. To increase the robustness of library generation when carrying out the Gel-Plus procedure, several steps in the protocol have been optimized and guidelines are provided.
The key advantage to using the TruSeq Nano DNA Library Prep kit is that only 100 ng of starting genomic DNA is required. This is increasingly important in studies that have limited amounts of DNA or where DNA needs to be split into multiple applications. In addition, the TruSeq Nano DNA Library Prep kit is a comprehensive solution containing all of the necessary reagents, barcodes, and size selection beads for convenient processing. The improved workflow includes bead-based size selection, therefore eliminating the time of gel-based methods. The kit also produces premier library quality, offering an excellent solution for studies requiring the highest coverage.
High-Resolution Melt (HRM) curve analysis was originally developed for SNP genotyping but has since been applied to different applications of mutational analysis. Applications for HRM include mutation discovery, DNA fingerprinting, species identification, HLA compatibility typing, allelic prevalence, and DNA methylation analysis.
Real-Time PCR has been used in quantification of gene expression, viral quantification, validation of array data, pathogen detection, and allelic discrimination.
Multiplex Real-Time PCR can be applied to relative quantification experiments where the gene of interest and reference gene are co-amplified in the same reaction. Multiplex Real-Time PCR can also be used for allelic discrimination assays, where two differentially labeled probes detect two alleles of a single nucleotide polymorphism. Another application of multiplex Real-Time PCR is pathogen detection, where multiple pathogens can be detected in one reaction.
Refer to the TruSeq Library Prep Pooling Guide for recommendations and guidelines for Illumina sequencing systems that require balanced index combinations.
Sequencing libraries prepared with the TruSeq DNA PCR-Free Sample Prep Kit requires the HiSeq X Reagent Kit v2. TruSeq DNA PCR-Free libraries are compatible only with workflow changes introduced with the v2 chemistry.
Reagent Kit Version
Control Software Version
TruSeq Nano DNA
Any HiSeq X kit version
Requires v3.0, or later
TruSeq DNA PCR-Free
Requires the HiSeq X v2 kit
Requires v3.1, or later
The HiSeq X Reagent Kit v2 requires HiSeq X Control Software v3.1, or later.
The PC Module has the same computing requirements as other GenomeStudio Microarray modules listed here.
Coverage and read depth requirements vary considerably depending on the form of methylation sequencing used. Please refer to the following references for specific recommendations:
|Sample Preparation||Quality DNA that has been chromatin immunoprecipitated with chemical modifications||Standard genomic DNA with no modifications|
|Enzymes / adaptors||Less is used||More is used|
|Product visible on gel||No||Yes|
|Polymerase||Standalone||Part of pre-mixed component|
|Quantitation of final product||Requires sensitive method like a BioAnalyzer||Less sensitive method required|
Although we strongly recommend using antibodies that have been shown to produce good results in ChIP-chip experiments, there are exceptions. ChIP grade antibodies were designed specifically for ChIP, but many also work well in other applications and assays. Many antibodies have been successfully tested in ChIP, so please consult directly with the antibody provider for more information. See more info.
All recipes use latest SBS chemistry but only v10 recipes enable dual indexing and eight-base barcodes for both single-index and dual-index runs.
Overloading the Nextera tagmentation reaction with more than 50 ng of genomic DNA leads to a larger library size distribution, which can lower the number of on-target sequencing reads. Less than 50 ng of input DNA leads to a smaller library size distribution. The result is reduced library diversity or elevated duplicate reads because many of these smaller library sized fragments would be eliminated during the size-selection procedures.
The HiSeq maintenance wash has three steps: a water wash, followed by a NaOH wash, and then a final water wash. You can expect the following delivered volumes from the eight lines of waste tubing:
50–500 ng of Total RNA, 11 µl at 50–500 ng/µl, according to Ambion Total Prep Kit #IL1791.
cRNA to load on chips: 750ng in 5 µl (150 ng/µl) for 8- and 12-sample, 1.5 µg in 10 µl (150 ng/µl) for 6-sample.
> 500 ng for two single-use activation reactions, 45* µl at 50–400 ng/µl.
*According to Zymo Bisulfite Conversion Kit D5001, D5002, D5004. Kit D5003 is not recommended because the elution volume is too large for this assay.
The TruSeq DNA PCR-Free Library Prep Guide provides guidelines for both a 350 bp and 550 bp average insert size based on paired-end sequencing results. The 350 bp workflow starts with 1 µg of gDNA, and the 550 bp workflow starts with 2 µg of gDNA.
You should maintain a lab temperature of 22°C ±3°C.
There are two major detection chemistries used for Real-Time PCR: enzymatic (NuPCR) and hydrolysis (TaqMan) probe-based chemistries, and DNA-binding SYBR Green I dye-based chemistry. Additional detection chemistries include Molecular Beacons, Scorpions probes and LUX primers.
NuPCR is a probe-based enzymatic chemistry consisting of amplification primers, DNAzyme oligonucleotides, and a universal substrate oligonucleotide. In the reaction sequence, two DNAzyme oligonucleotides (PartZyme A and PartZyme B) bind to the target DNA during annealing to create a NuZyme complex. A universal substrate oligo containing a 5´ fluorophore and a 3´ quencher then attaches to the complex, and PCR amplification primers attach to the DNA, flanking the target sequence on both sides. Detectable fluorescence is produced when the catalytic activity of the NuZyme complex separates the flourosphore and the quencher from each other on the substrate oligo. After the NuZyme complex is released from the target DNA, a copy of the target sequence is created when polymerase binds and extends the target sequence from the PCR amplification primers. NuPCR will continue to repeat the binding and separating process, producing amplification of the DNA and fluorescent signal.
In TaqMan probe-based chemistry, also known as the fluorogenic 5' nuclease assay, an oligonucleotide probe anneals to a specific sequence downstream of one of the PCR primers. The oligonucleotide is labeled with a fluorescent reporter dye at the 5' end and a quencher dye at the 3'. When the probe is intact, the reporter is in close proximity to the quencher and the fluorescent signal is low as the energy from the reporter will be transferred to the quencher through Fluorescent Resonant Energy Transfer (FRET). During PCR, as Taq DNA polymerase extends from the primers, the 5' exonuclease activity of the enzyme cleaves the annealed probe to separate the reporter dye from the quencher dye, increasing the fluorescent signal.
SYBR Green I is a dye that binds only to double-stranded DNA (dsDNA) and its fluorescent signal increases only when bound to dsDNA. During PCR, the fluorescent signal of SYBR Green I increases along with the dsDNA amplicon.
The controls that are required depend on the type of Real-Time PCR experiment:
The minimum system requirements are Windows 7 or later, 64-bit CPU, 2 GB RAM minimum, 4 GB recommended, and 25 MB hard drive space for installation.
Importing whole genome data or large gVCF files requires more RAM. For more information, see the entry in the File Formats section on this page, "What are the requirements for importing whole genome data or large gVCF files?".
There are three phases of PCR amplification: exponential, linear, and plateau. The exponential phase is the first phase of PCR amplification. Reaction components are in excess, there is an exact doubling of product each cycle, and the reaction is specific and precise. Real-Time PCR measures the Cq value at this phase of PCR. The linear phase is the second phase of PCR amplification. The reaction components are being consumed, amplification slows, and the reactions become highly variable. The final phase of PCR amplification is the plateau phase. The reaction is complete and no more products are being generated. Traditional PCR takes its measurements during this phase of PCR.
See the Cluster Densities Specifications technical bulletin on MyIllumina for recommendations.
The protocol has been developed and validated with 100 ng of high quality DNA for a 350 bp insert size and 200 ng of high quality DNA for a 550 bp insert size.
Importing large gVCF files requires more memory and takes longer to import. Use the following information as a guideline to estimate required RAM. This information is based on a Quad-Core Xeon® processor with 16 GB RAM.
For genotyping, Illumina requires at least 1.75 μg of pure and intact DNA per sample in 35 μl with a minimum concentration of 50 ng/ul as measured by PicoGreen. For methylation, Illumina requires at least 2.10 μg of pure and intact DNA per sample in 40 μl with a minimum concentration of 70 ng/ul as measured by PicoGreen. See the Illumina Genotyping Service Guidelines and the Illumina Methylation Service Guidelines for more information.
Illumina requires at least 2 μg of pure and intact DNA per sample. Refer to the Sequencing Service Process for more information.
For a dual flow cell 2x101 cycle run (200 Gb) on the HiSeq 2000 using HCS v1.3 and prior, you can expect 2 TB of intensity data (optionally transferred to a server), 250 GB of base call and quality score information, and 1.2 TB of space for alignment output not including 6 TB of disk space used for temporary files removed before completion of alignment. Using HCS v1.4 and Flow Cell v3, storage requirements for raw data are approximately 60% greater than current runs based on additional swath data and increased cluster density.
Storage requirements for raw data are approximately 60% greater than current runs based on additional swath data and increased cluster density.
There are no changes for MiSeq analysis. HiSeq and GA data require an upgrade to CASAVA 1.8.2 to demultiplex dual-indexed libraries. It is also recommended to upgrade to SAV 1.8.4 or higher to use the new Index tab for real time demultiplexing information.
If you are upgrading from an earlier version of software (i.e., HCS 1.3 or lower), please contact Technical Support as you may need to re-validate. Validation of HCS 1.5/RTA 1.13 should not be required if you are upgrading from HCS 1.4/RTA 1.12. The changes in RTA do not affect data quality and the changes in HCS were to update the user interface to enable dual indexing. Please refer to the HCS 1.5 Release Notes for additional information about new features in this software package.
Typical output concentrations from the pre-enriched library are 40–125 ng/μl.
Typical output concentrations from the final enriched library are (nM calculation assumes a 400 bp library size):
The quality and quantity of genomic DNA input into the Nextera tagmentation reaction will affect the library size distribution. A larger peak distribution (> 350 bp) can be indicative of > 50 ng genomic DNA input going into the Nextera tagmentation reaction. Conversely, a smaller sample peak distribution (< 225 bp) can be indicative of < 50 ng genomic DNA or fragmented, low quality genomic DNA.
It is critical to accurately quantify the concentration of input genomic DNA. Illumina recommends quantifying the starting genomic DNA using a fluorometric-based method specific to double-stranded DNA, such as QuantiFluor or PicoGreen. For more information, see the DNA Input Recommendations section of the Nextera Rapid Capture Enrichment Guide.
The MiSeq System uses the MiSeq Reagent Kit, which includes specially designed and packaged reagents for cluster generation and sequencing.
For kit compatibility information, see the Illumina Version Compatibility Reference.
In the demultiplexing workflow (configureBcltoFastq.pl), the --use-bases-mask parameter will need to be included if dual indexing is indicated in the sample sheet: Example: --use-bases-mask=Y*,I*,I* (for single-end runs) or --use-bases-mask=Y*,I*,I*,Y* (for paired-end runs).
Please see the CASAVA User Guide and release notes for additional user-input commands that are required in CASAVA.
A full list of user supplied items can be found in the Consumables and Equipment section of the Nextera Rapid Capture Enrichment Guide.
A full list of user supplied items can be found in the Consumables and Equipment section of the TruSeq Synthetic Long-Read DNA Library Prep Guide or TruSeq Synthetic Long-Read DNA Library Prep consumables and equipment excerpt. Comparable performance is not guaranteed when using alternate equipment.
A full list of user supplied items can be found in the Consumables and Equipment section of the TruSeq Targeted RNA Expression Guide. Comparable performance is not guaranteed when using alternate equipment.
A full list of user supplied items can be found in the Consumables and Equipment section of the ForenSeq DNA Signature Prep Guide or ForenSeq DNA Signature Prep consumables and equipment excerpt. Comparable performance is not guaranteed when using alternate equipment.
Use HiSeq X Control Software v3.1, or later, with the HiSeq X Reagent Kit v2. Version 3.1 is required to sequencing TruSeq DNA PCR-Free libraries.
Reagent Kit Version
Control Software Version
TruSeq Nano DNA
Any HiSeq X kit version
Requires v3.0, or later
TruSeq DNA PCR-Free
Requires the HiSeq X v2 kit
Requires v3.1, or later
To remove the least reliable data from the analysis results, often derived from overlapping clusters, raw data are filtered to remove any reads that do not meet the overall quality as measured by the Illumina chastity filter. The chastity of a base call is calculated as the ratio of the brightest intensity divided by the sum of the brightest and second brightest intensities.
Clusters passing filter are represented by PF in analysis reports. Clusters pass filter if no more than one base call in the first 25 cycles has a chastity of < 0.6.
The NextSeq 500 seamlessly streams data to BaseSpace, where demultiplexing and conversion to industry-standard FASTQ file formats occurs automatically as the final step in data transfer to Basespace. BaseSpace provides a number of analysis tools, including the Core BaseSpace Apps for whole genome sequencing, enrichment, and RNA-Seq.
Illumina provides the bcl2fastq 2.0 conversion software for demultiplexing and conversion of NextSeq 500 output to standard FASTQ file formats. This conversion software enables analysis of NextSeq 500 data in any third-party NGS analysis solutions.
For each sample, the following information is required: SampleName, SampleDescription, Project, i5Index, i7Index SampleType. See the ForenSeq Universal Analysis Software Guide for more details on these fields.
The ChIP-seq workflow generates demultiplexed FASTQ files.
Two data compression options are zipping of BCL files and binning of Q-scores. Other run folder files are unchanged. These options are available during run setup in HCS v2.2. If you are using BaseSpace for data storage and analysis, BCL files are zipped automatically. Due to the size of the run folder with the extra cycles and shorter run durations, zipped BCL files are required for HiSeq v4 runs. This setting cannot be turned off. You can select or deselect Q-score binning depending on your preference.
You can expect greater than 75% of bases above Q30 with a 2 x 150 bp run using the following libraries:
These are supplied by the submitter of the data. Samples may have been used as part of the case group in an experimental design (e.g., a sample from a diabetes patient). In this instance, "diabetes" may be reflected in the "Positive Phenotype" for this sample. These samples are still valuable to other studies that are not looking at this Positive Phenotype, and in fact it may be very valuable to obtain accurate population representation in control sets. Similarly, "Negative Phenotype" is supplied by the submitter and is intended to indicate which phenotypes the sample has been screened for and are negative. Please note that some submitters have chosen also to use the Phenotype field to note the sources of the samples (e.g., blood, lymphocytes). In addition, the entry "HapMap" indicates HapMap samples from Illumina.
Longer drying times may be required depending on environmental variables and the amount of ethanol remaining in the well. However, take care not to over-dry beads as this can impact sample recovery.
The Eco analysis software does not provide alternative methods of relative quantification analysis, but third-party tools are available. You can export all data in *.csv format from the Illumina Eco analysis software and import the data into third-party analysis software such as REST or qBase.
VariantStudio needs external connections to annotate. For annotation, VariantStudio simply needs access to the annotation URL via port 80. This URL can be found in the installation folder, in the file VariantStudio.exe.config, next to the key annotationServiceUri. This link should be an Amazon Web Services or Illumina link. In addition to the annotation connection, VariantStudio must make a one-time connection to basespace.illumina.com and icom.illumina.com, also through port 80. This connection serves to make sure that the user has access to the Illumina Annotation Service. This connection is needed again if the Forget BaseSpace Logon option is used, or if the BaseSpace account information is not saved.
A = All isoforms. The probe is designed to hit all splice isoforms of a gene.
I = Isoform specific. The probe is designed to hit a specific splice isoform of a gene, for which multiple isoforms are known to exist.
S = Single isoform. The gene has only one known splice isoform and our probe hits it.
M = Multiple isoforms. This gene has multiple isoforms. The probe targets more than one and fewer than all of them.
Please refer to our technical note entitled “TOP/BOT” Strand and “A/B” Allele (PDF).
The ForenSeq Universal Analysis Software is used to create runs, monitor runs, review results, and align reads to make allele and genotype calls for sample information. Optionally, the software can generate reports, population statistics, sample comparisons, phenotype estimations and bio-geographical ancestry estimations.
This score reflects the predicted relative performance of amplicons given Illumina's evaluation of the amplicon probes. It takes into account the target specificity, GC content, and other design parameters.
The following table details the excitation and emission wavelengths of Eco in each channel. The emission filters combined with spectral de-convolution algorithms effectively minimize cross-talk between dyes. Eco is factory calibrated for SYBR Green I, FAM, HEX, VIC, ROX, Cy5, and Q670. You can use other dyes if they are within the wavelength range of the emission filters.
Channel Excitation (nm)
Example Fluorophores Detected
SYBR Green I, FAM
In VariantStudio, you can export information displayed in the Variants table, which includes variants and associated annotations, in a TSV file format. Sample reports are generated in either RTF or PDF file formats.
TruSeq ChIP libraries can be sequenced using either Single Read or Paired End flow cells.
The HiSeq X system uses patterned flow cell technology. Reagents provided in the HiSeq X Reagent Kit are designed and optimized for the HiSeq X flow cell. Non-patterned flow cells and SBS kits designed for other HiSeq models have not been validated and are not supported for use on the HiSeq X system.
There are multiple, published forms of NGS-based methylation, each of which offers its pros and cons. We strongly recommend consulting recent review articles offering comparative analysis of multiple applications. A few examples are given below:
In the event that data transfer is interrupted during a run, data are stored temporarily on the instrument computer until the connection is restored. When the connection is restored, transferring of data resumes automatically.
If the connection is not restored before the end of the run, data must be removed from the instrument computer manually before a subsequent run can begin.
The Illumina FastTrack Microarray Services lab repeats all samples that have adequate concentrations but low genotyping quality. This steps makes sure that each sample is given at least two genotyping attempts to optimize data quality. There is no charge for second attempts, but you are charged for all samples attempted.
An account can be set up on the ForenSeq Universal Analysis Software. See the ForenSeq Universal Analysis Software Guide for more details. This account is used on the sequencing instrument to log in to MiSeq FGx Control Software and start the runs created by the account holder on the ForenSeq Universal Analysis Software.
Cleanup procedures have only been optimized and validated using the magnetic stand specified in the appropriate TruSeq DNA or RNA library prep guide. Comparable performance is not guaranteed when using other magnets. Other magnets can be used, however you may want to test how long samples need to sit on the magnet, as times may vary from the protocol.
The Eco system completes 40 PCR cycles in about 40 minutes, so you can perform multiple runs quickly. The Eco system's superior uniformity provides excellent sample reproducibility and can reduce the number of technical replicates that are required for your experiment. And since it is a personal Real-Time PCR system, you should have access to the instrument virtually any time you need it.
Although the TruSeq Nano DNA and TruSeq DNA PCR-Free library prep kits are compatible with dual indexing, only 8 bp single indexing is supported on the HiSeq X system.
Real-Time PCR uses various fluorescent detection chemistries that allow you to monitor the PCR reaction as it progresses. The amount of fluorescent signal generated is directly proportional to the amount of DNA being synthesized during the PCR reaction. Data are collected at each cycle as opposed to traditional PCR, which collects data at the end of the reaction. This allows samples to be characterized by when amplification is first detected as opposed to the amount of product generated after PCR cycling. The greater the amount of the target sequence, the earlier amplification will be detected.
The ForenSeq DNA Signature Prep Kit is used to determine which STRs and SNP genotypes are present in an individual sample.
HRM is a recent advancement to the traditional melt curve analysis that significantly increases the amount of detail and information that can be captured. It is sensitive enough to be able to differentiate sequence differences within PCR amplicons down to a single nucleotide. Mutations within amplicons are detected as either a shift in the Tm of the product or a change in the shape of the melting curve.
For information on Illumina's regulatory, quality, and instrument safety certifications, including ISO certifications, go to the Regulatory and Quality Information page.
TruSeq Targeted RNA Expression is a library prep workflow that enables efficient, multiplexed gene expression profiling for 12–1000 targets per sample and up to 384 samples in a single MiSeq run.
The manifest folder contains manifest files required for the Custom Amplicon and PCR Amplicon workflows. A manifest file is required to specify the alignments to a reference and the targeted reference regions used in the workflow. You can specify the location of the manifest folder using the MiSeq software interface. Before you begin the sequencing run, copy the manifest file to the specified location.
Clusters passing filters are those clusters that pass a number of sequencing quality thresholds. % passing filter is calculated over the first 25 cycles of sequencing, and only clusters that pass filters are included in the final analysis.
Manifest files contain a list of genomic regions and coordinates being targeted for enrichment in the Nextera Rapid Capture Exome and Expanded Exome kits. They are also required in the data analysis process for alignment and variant calling in targeted regions. Each product contains a _targeted regions and _probe manifest file.
Nextera Rapid Capture Exome and Expanded Exome Enrichment manifests can be downloaded from the Nextera Rapid Capture Exome Enrichment Downloads support page.
A patterned flow cell is a flow cell with billions of ordered nano-wells that are manufactured into the flow cell glass. The ordered wells allow for the generation of sequencing clusters in an ordered arrangement. Clusters are aligned more closely together increasing the number of output reads and amount of sequence data generated. The HiSeq X patterned flow cell contains 8 lanes and has the same general dimensions as a HiSeq high-output flow cell.
The bcl2fastq 2.0 software package converts base call (BCL) files generated on the NextSeq 500. If you plan to use a third-party data analysis solution outside of BaseSpace, set the NextSeq to operate in Standalone mode and use the bcl2fastq 2.0 conversion software. For more information, see the bcl2fastq Conversion User Guide v2.0.
Although bcl2fastq 2.0 is similar to bcl2fastq 1.8.4, only version 2.0, or later, is compatible with NextSeq data. Use version 1.8.4 to convert data from HiSeq or MiSeq.
The bcl2fastq v1.8.4 conversion software is a separate piece of standalone software that is run on a Linux scientific computing system. The installer can be downloaded from the Illumina website. System requirements are outlined in the bcl2fastq User Guide (part # 15038058). If BCL files are zipped, then the use of the bcl2fastq v1.8.4 is required.
Melt curve analysis is a post-PCR analysis that is compatible with SYBR Green I-based assays. During a melt curve, amplicons produced during PCR are dissociated (or melted) by slowly ramping the instrument from a low temperature to high temperature. The fluorescence is monitored throughout. As the amplification products transition from dsDNA to single-stranded DNA and the SYBR Green I is no longer bound, there is a sharp decrease in fluorescence. The midpoint of this transition is known as the melting temperature (Tm) of the sample and is characteristic of a given DNA sequence. Melt curves are useful for determining the specificity of a PCR reaction, as any non-specific amplification products will have a different melt curve profile than the target sequence.
Multiplex Real-Time PCR is a technique in which multiple target sequences are amplified and detected in a single PCR reaction. Amplified sequences are distinguished by the use of different dyes conjugated to the TaqMan probes. The number of targets that can be detected in a single reaction is technically limited only by the availability of spectrally distinct dyes and the ability of the Real-Time PCR instrument to effectively excite and detect those dyes. Some advantages of multiplex Real-Time PCR are reduced reagent costs, reduction in sample use, and increased throughput.
The new Nextera Mate Pair kit offers several advantages over the previous Mate Pair kit: significantly less DNA input (1-4 ug as compared to 10-20 ug for the original kit), a faster and simplified workflow, and a higher supported read length of 2x250.
Other advantages of the Nextera Mate Pair kit include utilization of a transposome-mediated fragmentation and adapter tagging of genomic DNA, an identifiable Mate Pair junction sequence, TruSeq DNA Sample Prep master-mixed reagents to reduce reagent containers and pipetting steps, TruSeq DNA Sample Prep adapter indexing compatibility with 12 indexes included in the kit, and a choice of two protocols: a Gel-Free Protocol and a Gel-Plus Protocol.
Phasing and prephasing occurs when a strand within a cluster falls behind or ahead respectively of the incorporation cycle. The % metric given shows an estimate of the fraction of strands that are phased or prephased. Low percentages indicate good run statistics.
Developing multiplex Real-Time PCR assays can be difficult and time-consuming. As the reaction complexity increases, significant optimization may be required to generate reliable data. It can be a challenge to develop multiplex assays that amplify all targets with equal efficiency.
When developing multiplex Real-Time PCR assays, you need to consider primer design, the relative expression levels of target sequences, and master mix / reagent conditions.
Use the same design criteria for each primer/probe set and screen all sequences against each other to determine any potential primer-dimer formation. In addition, perform a BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine primer specificity.
If the expression levels of the target sequences are significantly different, the most abundant target will be preferentially amplified and deplete all the reaction components, compromising amplification of the less abundant targets. One way to address this issue is to limit the primer concentrations of the most abundant target, using the lowest primer concentration that produces the same Cq and PCR efficiency. Limiting the primer allows the most abundant target to amplify and go to completion without depleting all the reagents needed for the other sequences.
Amplifying multiple target sequences creates additional demand for reaction components. Taq DNA polymerase, Mg++ and dNTP concentrations may need to be optimized to improve amplification of all targets. Master mixes optimized specifically for multiplex Real-Time PCR are now commercially available, and can reduce the amount of time required for optimization.
Compare experiment data that uses a single assay with the experiment data when the assay is combined into a multiplex assay. Sensitivity and PCR efficiency needs to be the same in both types of assay use.
High-Resolution Melt (HRM) curve analysis requires a different class of dsDNA binding dyes, extremely precise instrumentation, and specialized software.
HRM analysis is generally performed using dsDNA binding dyes other than SYBR Green I. These dyes are known as saturating dsDNA-binding dyes, and include SYTO 9 (Invitrogen), LCGreen (Idaho Tech), and EvaGreen (Biotium Inc.). These dyes differ from SYBR Green I in that they are significantly less inhibitory to PCR. This reduced inhibition allows them to be used at much higher concentrations than SYBR Green I that saturate the dsDNA amplicons. Greater dye saturation provides greater sensitivity and resolution of melt curve profiles.
Extremely precise instrumentation is important for HRM. Since some mutations only cause Tm shifts of a fraction of a degree, any thermal or optical non-uniformity will reduce the ability to detect these sequence changes. To be able to perform HRM analysis an instrument needs to have a fast acquisition rate, precise temperature control, and an absolute minimum of thermal and optical variation between samples.
HRM also requires software with specialized analysis algorithms that can analyze the shape of melt profiles and group similar melt profiles together. HRM data can be viewed as either normalized melt curves or difference plots. Difference plots show the difference in fluorescence from a selected reference sample. Some software also features an auto-call feature, which can automatically assign genotypes based on melt profiles.
You might notice an intermittent clicking sound coming from the right side of the instrument. This is normal. The clicking sound indicates that the refrigeration condensation pump is working. Under normal conditions, the sound occurs for about five minutes in a 12-hour period.
The Diff Score is a transformation of the p-value that provides directionality to the p-value based on the difference between the average signal in the reference group vs. the comparison group. The formula is: DiffScore = 10*sgn(µcond-µref)*log10p; For a p-value of 0.05, DiffScore = ± 13; For a p-value of 0.01, DiffScore = ± 22; For a p-value of 0.001, DiffScore = ± 33 The p-value column is hidden by default. To display this column, use the Column Chooser.
HiSeq Analysis Software provides rapid and easy alignment and variant calling for whole human genomes or libraries prepared with the Nextera Rapid Capture exome enrichment kit. For Whole Human Genome Sequencing, HiSeq Analysis Software features the Isaac analysis workflow, which is an ultra-fast and accurate sequence analysis software, providing a 4–6 times speed increase over existing methods. For Nextera Rapid Capture analysis, the BWA alignment and GATK variant calling methods are used. The software can be run through the command line or through a graphical user interface package called Analysis Visual Controller Software (AVC).
The Infinium OncoArray-500K is a custom iSelect BeadChip, designed by the OncoArray consortium to assess cancer predisposition and risk. It is the next generation of the groundbreaking iCOGs array. The OncoArray-500K was designed to allow thorough characterization of the genetic architecture of susceptibility for five common cancers (breast, colorectal, lung, ovarian, and prostate).
Illumina found that separating the enzyme from the buffer provides a minimum of a nine month shelf life from date of manufacture. The MCS3 had a four month shelf life. As a result, the MCS3 has been split into two tubes containing MCS4 (buffer) and RTE (Reverse Transcriptase Enzyme).
The GenomeStudio Polyploidy Clustering Module (PC Module) can identify clusters for samples where the standard diploid clustering algorithm is inappropriate or not useful, such as for polyploidy organisms like wheat and potato.
The acquisition time for all four channels is approximately six seconds.
TruSeq SBS v3 reagents enable an alternative workflow for loading all SBS reagents at the start of a 2x101-cycle sequencing run for both Read 1 and Read 2. Using this workflow might result in a slight increase in phasing in Read 2, which should not result in a decrease in quality.
KaryoStudio provides several items for you to examine when you are QCing data. The LogRDev metric provides a measure of noise in the intensity data, and is essentially a measure of standard deviation of Log R values across the autosomes. The "percent aberration" metric is a sum of all of the found regions in a sample divided by the entire length of the genome. In a blood sample, where you expect to have little to no aberrations, you will see a very small (<1%) measure for % aberration. In cases where you have a higher number, it may indicate a sample processing issue. Both of these metrics can be impacted by real biological variation in samples, so they should be examined holistically while taking into account the data viewed in the IGV.For specific troubleshooting issues and access to the controls dashboard, load your data into GenomeStudio, if you have access to that software. Otherwise, contact technical support with any additional questions on troubleshooting your data.
The TruSeq Nano DNA Library Prep Guide contains both a Low Sample (LS) and High Sample (HS) protocol. These protocols differ in the types of plates used and the method of incubation and mixing. The LS protocol uses 0.3 ml PCR plates, the incubation steps are done on a thermal cycler, and mixing method is pipetting. The HS protocol is done in MIDI plates, requires additional equipment such as microheating system for all incubation steps, and mixing method is done on a microplate shaker. The LS protocol is optimized for processing 24 samples at a time and HS protocol for more than 24 samples. Both the LT and HT kits can be used with either the LS or HS protocol and will give comparable results; however, the HS protocol can yield more consistent results between samples.
Call Rate is carried over from the Samples Table in the GenomeStudio Genotyping Module in which the original genotyping project (.bsc) was created. Entries in the Call Rate column do not change when SNPs are clustered in the PC Module. In contrast, the Poly Call Rate is calculated from clustering SNPs in the PC Module and represents the percentage of SNPs for which a given sample was assigned to a cluster.
DNA Primer Mix A—Contains primer pairs for 59 STRs (including 27 autosomal STRs and 8 X and 24 Y haplotype markers) and 95 identity-informative SNPs.
DNA Primer Mix B—Contains all markers in DNA Primer Mix A, plus primer pairs for 56 ancestry-informative SNPs and 22 phenotypic-informative SNPs (two ancestry-informative SNPs are also used for phenotype prediction).
Nextera Rapid Capture Enrichment is an updated, faster enrichment workflow that offers a more efficient exome (37 Mb) in addition to the more comprehensive existing exome product (62 Mb).
The Nextera Rapid Capture Enrichment kits include similar components as the Nextera Enrichment kits, however formulations have changed for several reagents in the Nextera Rapid Capture Enrichment kits. The enrichment workflow has been shortened by reducing enrichment hybridization time and utilizating more efficient wash steps with an improved washing buffer.
The Ribo-Zero Human/Mouse/Rat kit depletes samples of cytoplasmic ribosomal RNA. The Ribo-Zero Gold kit depletes samples of both cytoplasmic and mitochondrial ribosomal RNA.
A relative quantification (also known as comparative quantification) assay quantifies changes in gene expression relative to a reference gene and reference sample. An absolute quantification assay uses a standard curve of known quantities to determine the quantity of unknown samples. Relative quantification results report fold changes in expression relative to the reference gene and reference sample. Absolute quantification results are reported as an absolute quantity (copies, ng, pg, etc.) extrapolated from the standard curve.
The difference between one-step and two-step real time RT-qPCR lies mainly in the reverse transcription step. In one-step RT-qPCR, a short reverse transcription (5–30 minute) reaction is followed by a PCR reaction in a single tube. In two-step RT-qPCR the reverse transcription reaction takes place in a separate tube. Each method has advantages and disadvantages, depending on the application.
One-step real time RT-qPCR, which uses gene-specific primers, is useful when analyzing a few genes over a large number of samples. Since both the RT and PCR reactions occur in the same tube, there is less pipetting and sample manipulation, possibly reducing variation and potential contamination. One-step RT-qPCR might not be as sensitive as two-step since its reverse transcription step is much shorter. The reaction conditions needed to support both the RT and PCR reactions might not be optimal for either reaction. Another drawback of one-step RT-qPCR is that it is not possible to archive the cDNA produced during the reverse transcription reaction.
Two-step real time RT-qPCR is useful when analyzing a large number of genes over a few samples. It supports a flexible priming strategy, allowing for oligo-dT, random primers, or gene-specific primers. Two-step RT-qPCR is generally more sensitive than one-step since the RT reaction is much longer and the RT and PCR reactions occur separately, meaning that they can be optimized individually. Also, the cDNA produced is more stable than the initial RNA sample and can be more easily archived for future use.
The Nextera Rapid Capture Expanded Exome Enrichment utilizes the same capture oligos as the TruSeq Exome Enrichment Kit paired with the new Nextera Rapid Capture Enrichment reagents.
Exons, UTR, miRNA
The TruSeq DNA PCR-Free LT Library Prep Kits come with adapter index tubes recommended for preparing 24 or less samples at a time. The LT kits come in two sets, A and B, and each set contains 12 unique indices for a total of 24 unique single-index adapters if both kits A and B are combined. Each kit (LT kit A or LT kit B) come with reagents sufficient for 24 samples. The TruSeq DNA PCR-Free HT Library Prep Kit comes in a 96 well adapter plate format with 96 dual-indexed adapters and enough reagents for 96 samples.
The TruSeq DNA PCR-Free Library Prep Guide contains both a Low Sample (LS) and High Sample (HS) protocol. These protocols differ in the types of plates used and the method of incubation and mixing. The LS protocol uses 0.3 ml PCR plates, the incubation steps are done on a thermal cycler, and mixing method is pipetting. The HS protocol is done in MIDI plates, requires additional equipment such as microheating system for all incubation steps, and mixing method is done on a microplate shaker. The LS protocol is optimized for processing 24 samples at a time and HS protocol for more than 24 samples. Both the LT and HT kits can be used with either the LS or HS protocol.
The TruSeq Nano DNA LT Library Prep kits come with single-index adapter tubes recommended for preparing 24 or less samples at a time. The LT kits come in two sets, A and B, and each set contains 12 unique single-index adapter tubes for a total of 24 unique single-index adapters if both kits A and B are combined. Each kit (LT kit A or LT kit B) come with reagents sufficient for 24 samples. The TruSeq Nano DNA HT Library Prep Kit comes in a 96 well adapter plate format with 96 dual-indexed adapters and enough reagents for 96 samples.
For the Phasing workflow—
For the Long-Read workflow—
The TruSeq sample prep high sample (HS) protocol requires additional peripheral equipment, but reduces user touch-points and is ideally suited for projects that include more than 48 samples prepared at one time. The low sample (LS) protocol requires minimal peripheral equipment, but requires more user manipulation and is best suited for projects that include 48 or fewer samples. For specific differences in the workflows, including required equipment, refer to the user guide for the TruSeq sample prep kit that you are using.
TruSeq high throughput (HT) sample prep kits each include reagents for 96 samples divided equally between two tubes. Each kit also includes an adapter plate containing 96 unique dual-indexes.
The TruSeq low throughput (LT) sample prep kits (A and B) each include reagents for 48 samples and 12 of 24 total adapters.
The length of the HT and LT adapters differ. Please see MyIllumina Support Bulletins for the sequences.
There is only one minor protocol change in that the amount of AMPure XP beads has been adjusted for the PCR clean-up for the HT kit. This has been optimized for the longer adapters present in the adapter plate. Please see each kit's user guide for more information.
TruSight One is a focused sequencing panel that enriches sample libraries for a selection of coding exons associated with human disease, capturing ~12 Mb of genomic content. The NRC Exome kit targets >98% of coding human coding exon content and captures ~37 Mb of genomic content. The NRC Expanded Exome panel includes NRC exome content and additional UTR, promoter, and miRNA targets with ~62 Mb genomic content. TruSight One and the Nextera Rapid Capture Exome and Expanded Exome kits all use Illumina Rapid Capture library prep technology.
The Open Source version of Isaac includes the component algorithms for the Isaac aligner and Variant Caller and is intended for developers. This version is not commercially supported and is provided as is under Illumina Open Source Software License available here.
For the majority of Illumina customers, we recommend using Isaac as provided as part of the HiSeq Analysis Software package.
HiSeq Analysis Software provides rapid and easy alignment and variant calling for Whole Human Genomes (using the Isaac component algorithms) or libraries prepared with the Nextera Rapid Capture (NRC) exome enrichment kit (using the BWA/GATK component algorithms). The software can be run through the command line or through graphical user interface package called Analysis Visual Controller Software (AVC). HAS is freely available, easy to install (rpm) and commercially supported. For more information, see our Support page. http://support.illumina.com/sequencing/sequencing_software/hiseq-analysis-software.ilmn
The peak power of the instrument is 500 VA. Typical power consumption during a run is around 180 VA.
Based on the latest advances in Illumina's reversible terminator SBS chemistry, the MiSeq System is the most accurate sequencing instrument available, providing the world's highest output of perfect, error-free reads. For examples of MiSeq performance, see the MiSeq publications page.
While coverage depth of individual target regions may vary, all targeted regions should be represented in the final enriched library.
A very low percentage of targets may not be represented in the final enriched library due to complex genomic regions, low probe specificity, skewed GC content, and other technical factors inherent to probe based enrichment and SBS. DesignStudio includes Design Warnings to highlight probes with an elevated risk of underperformance.
r2 between BeadChips: .99
r2 within BeadChip: .99
The size of the analysis output folder for each sequencing run depends on the number of cycles in your run. Typically, a 150-cycle paired-end run (2 x 151 cycles) generates approximately 3 GB in output.
1 µg of high quality gDNA is required for final libraries with an average 350 bp insert size and 2 µg of high quality gDNA is required for a 550 bp insert size.
When samples arrive at the Illumina Microarray Services lab, the microarray team quantifies the samples using PicoGreen quantification. Samples proceed through the Illumina genotyping or methylation workflow, which is fully automated and integrated with Illumina LIMS to ensure positive sample tracking. After completion of every project, a project manager assesses the sample quality and marker performance to identify those samples or markers that do not perform well. For genotyping projects, genotype calls are optimized through cluster editing and reports are created to be sent as part of the project deliverables.
When samples arrive at the Illumina Sequencing Services lab, the sequencing team quantifies the samples using PicoGreen quantification. Samples proceed through library preparation in the Illumina Pre-PCR laboratory. Then, samples proceed to the Post-PCR laboratory, where the libraries go through clustering and sequencing. For more details on the lab workflow, see the .
Using NextSeq Control Software v1.3, the library loading volume and library concentration are lower than with earlier versions of the software. The loading volume is 1.3 ml and the loading concentration is 1.8 pM.
With NextSeq Control Software v1.2, and earlier, the library loading volume is 3 ml and the loading concentration is 3 pM. However, the loading concentration can vary depending on library preparation and quantification methods.
For more information, see Denaturing and Diluting Libraries for the NextSeq 500 (15048776).
The final library should show a size distribution from ~250–300 bp.
The fluid contained in the heating block is specifically designed for use in heat transfer applications and has been used for the last 30 years as an electronic coolant. The fluid is hermetically sealed in each block, is environmentally benign, and has a low vapor pressure.
OPTICS is an acronym for "Ordering Points to Identify Clustering Structure". It is a sub-algorithm of DBSCAN, developed to be more robust to changes in input parameters. This trait makes OPTICS more suited for initial clustering. DBSCAN is an acronym for "Density-based Spatial Clustering of Applications with Noise". This algorithm is more sensitive to initial input parameters such as cluster distance. DBSCAN is more suited for differentiating clusters that are very close together, and should typically applied to SNPs for which OPTICS does not yield satisfactory results.
If you are using the TruSeq Custom Amplicon Kit, 96 samples can be pooled in a single run combining up to 384 amplicons per sample.
For best results, Illumina recommends small genome (up to 20 Mb) sequencing on the MiSeq.
This varies for each user depending on the type of cytogenetics lab in which the samples are analyzed. For example, some cytogenetics labs tend to look at all aberrations greater than 250kb in size. Others may look at aberrations 100kb or greater in size. Therefore, it is up to the user to decide.
You may want to set a threshold for the minimum number of SNPs per region, such as 50. This information can be found in the # SNPs column in the Found Regions table.Remember that more aberrations appear in the Found Regions table when you allow for regions of a smaller size. In this case, many of the regions may not yet be linked to a specific phenotype. Therefore, users may want to try a "top-down" approach, beginning with the largest regions that may be most likely already linked to a phenotype. Depending on the product in use, you may find aberrations as small as 1kb in size.
Illumina recommends a minimum of 50 ng total RNA. This generally produces 5-10 ug of amplified RNA, which should be sufficient for 10-20 arrays.
The Eco system is extremely quiet, producing less than 62 db during a run.
The recommended raw cluster density is between 800 K/mm2 and 1.4M M/mm2. Depending on the number of samples being run at one time, it is possible to get ample reads/sample at < 800 K/mm2. Cluster densities above 1.4M M/mm2 raw are likely to affect sequencing quality and therefore advised against.
The recommended maximum cluster density is 750,000-850,000 clusters/mm² when using Illumina's v3 cluster generation and sequencing reagents in combination with HCS v1.4.
The NextSeq 500 generates base call (BCL) files aggregated by lane with one BCL file for each lane, for each cycle. The aggregated file contains the base call and associated quality score for every cluster for that lane. For each lane, one base call index file lists the original tile information in a pair of values for each tile.
When using BaseSpace, BCL files are automatically converted to FASTQ files when data transfer is complete. If BCL files are saved to a local server, use bcl2fastq 2.0 to convert base calls from a NextSeq run. This FASTQ converter must be run on a Linux server.
The output is greater than 7 Gb (2 x 250 bp read length) and greater than 15 million reads/tags (based on cluster density). For more information, see the MiSeq System Product Information Sheet.
The arrays listed below have the following overlapping SNPs:
The human sequencing control acts to make sure that the sequencing run is performing as expected. If there is an issue with the run, the human sequencing control provides information as to whether the problem is related to the instrument or library. The human sequencing controlalso acts to allow the run to complete successfully, where degraded or low quality libraries do not have the long STR sequences required to last through 351 cycles.
You can perform up to 2 x 250 bp, or 500 cycles of sequencing on the MiSeq using the MiSeq Reagent Kit v2. However, as few as 36 cycles can be used for some applications.
Using the MiSeq Reagent Kit v3, you can perform up to 2 x 300 bp, or 600 cycles of sequencing. MiSeq Reagent Kit v3 is available in two sizes, 600 cycles and 150 cycles.
When quantifying the post-enriched library using a fluorometric method, clustering at 12.5 pM generates cluster densities ranging from 1,200 k - 1,400 k clusters/mm2 using the MiSeq v3 software and reagents. Results vary based on your method of quantification and instrument-to-instrument variability. Illumina recommends that you determine the library concentration to cluster density relationship based on your lab instrumentation.
|Rapid (2 Lane)||High Output (8 Lane)|
|Recommended Cluster Density||750-900 k||750-800 k|
|Clusters per lane (PF)||130-150 M||187-210 M|
|Per flow cell output (2 x 100 bp)||50-60 Gb||300 Gb|
|Per flow cell output (2 x 150 bp)||75-90 Gb||No supported|
|Bases >Q30 (2 x 50 bp)||85%||85%|
|Bases >Q30 (2 x 100 bp)||80%||80%|
|Bases >Q30 (2 x 150 bp)||75%||Not supported|
Illumina recommends 5–10 ng of ChIP-enriched, fragmented DNA.
The supported insert size on the HiSeq X system is 350 bp for TruSeq Nano DNA and TruSeq DNA PCR-Free libraries.
Illumina recommends 2 x 100 bp runs.
The TruSight One assay is designed to be sequenced with a 2X151 cycle paired-end sequencing run to maximize coverage of the targeted region. The design also minimizes the likelihood of sequencing into uninformative adapter sequence. TruSight One libraries use dual indices and additional 8 cycle index 1 and 8 cycle index 2 reads are necessary to demultiplex pooled enrichments. Consult the TruSight One User Guide for additional details on sample pooling and dual-indexed sequencing.
The read length and format (single read versus paired end) are important considerations in the design of RNA sequencing experiments. The table below provide guidance on some factors to consider. These recommendations are based on internal and external data, but do not represent strict cut-offs. Needs for individual projects may vary based on multiple variables as well as user preference.
Read Type (bp)
Read Depth/Sample (mRNA/Total RNA)
Gene profiling (gene-level counts)
1 x 50
>5 m / >10 m
Discovery (alternative transcripts, gene fusions, etc.)
2 x 50 - 75
≥50 m / >100 m
Complete transcriptome annotation
2 x 75 - 100
≥100 m / ≥200 m
Illumina recommends a DNA insert size range of 200–800 bp.
One year from the date of manufacture. The kit will contain an expiration date on the label. Illumina guarantees at least 3 months from the date of receipt. This is the same as the TruSeq DNA LT and TruSeq RNA v2 kits.
Expiration dates are printed on the kit box labels and reagent tubes. Illumina guarantees 3 months of shelf life from the time of kit shipment.
Expiration dates are printed on the kit box labels and reagent tubes. Illumina guarantees 3 months of shelf life from the time of kit shipment.
Our policy for the VariantStudio software is to support the previous software version for 12 months after a new version is released. The content of the Annotation Database is aggregated from a broad range of external sources, including public and private sources. At the time of a release for the Annotation Database, the content is locked down and versioned. By the nature of private annotation sources, our ability to maintain annotation content for the 12-month period may not be possible in rare occurrences. In these rare occurrences, we make our best efforts to communicate the changes to you as soon as possible to reduce any potential impact on your pipeline processes.
The temperature resolution of the Eco system is 0.1° C.
Throughput is dependent upon the level of multiplexing and whether you are running a single-color or dual-color detection scanner. Typical throughputs are:
Each system can generate 1.6–1.8 Tb in less than 3 days with greater than 75% of bases about Q30 from a 2 x 150 bp run. This throughput enables 16 genomes covered at 30x per run per system. For more information, see the HiSeq X system performance specifications page.
Turnaround time depends on the selected product and the requested number of samples. Turnaround time also depends on when Illumina receives samples. See the Sequencing Service Process page for estimated turnaround time. Contact your local account manager or sales representative or submit questions through the Illumina website
Turnaround time depends on the selected product and the requested number of samples. Turnaround time also depends on applicable loci selection and on when Illumina receives samples. See the Microarray Service Process page for estimated turnaround time. Contact your local account manager or sales representative or submit questions through the Illumina website.
The size of the library distribution of DNA fragments ranges from approximately 200 bp–1 kb, with a final product expected at approximately 260 bp. A larger fragment size is expected for good FFPE RNA (> 350 nt), while a smaller fragment size is expected for poor FFPE RNA. The following is an example:
Nextera Rapid Capture Enrichment libraries generally range from about 150–1000 bp with the main peak at about 300–350 bp. The following is an example of a post-enrichment (12-plex enrichment) library distribution:
See the appropriate HiSeq instrument user guide for details on the loading of reagents with different workflows and which primers you need to use for your library type.
You can export data displayed in the Found Regions table as either a single row or the entire table.
There are no other reporting functions available in KaryoStudio.
A quality score (or Q-score) is a prediction of the probability of an incorrect base call. Based on the Phred scale, the Q-score serves as a compact way to communicate very small error probabilities. Given a base call, X, the probability that X is not true, P(~X), is expressed by a quality score, Q(X), according to the relationship:
Q(X) = -10 log10(P(~X))
where P(~X) is the estimated probability of the base call being wrong.
A quality score of 10 indicates an error probability of 0.1, a quality score of 20 indicates an error probability of 0.01, a quality score of 30 indicates an error probability of 0.001, and so on.
During analysis, base call quality scores are written to FASTQ files in an encoded compact form, which uses only one byte per quality value. This method represents the quality score with an ASCII code equal to the value + 33.
We provide the same support as for standard genomic DNA resequencing. However, we do not provide support on antibodies or the ChIP portion.
NextSeq 500 kits are available in three sizes: 300 cycles (2 x 150), 150 cycles (2 x 75), and 75 cycles (1 x 75). For output specifications, see NextSeq 500 specifications page.
TruSeq Small RNA Library Prep kits currently support 48-plex per lane.
Up to 96-plex indexing is supported for TruSeq Custom Amplicon, Nextera, Nextera XT, and TruSeq DNA Sample Prep Kits.
There are 96 available barcodes using 12x8 indices, combining 12 Index 1(i7) with 8 Index 2 (i5) indices.
TruSeq Exome Enrichment supports pre-enrichment pooling of up to six samples. Please refer to the TruSeq Exome Enrichment Data Sheet for additional information.
The Illumina Eco analysis software uses the ΔΔCq method (Livak et al., 2001), which is the most commonly used analysis method for relative quantification. The ΔΔCq method reports fold change in gene expression of a target gene relative to that of a reference gene and reference (calibrator) sample. The reference gene is typically a housekeeping gene such as GAPDH, β-actin, β2-microglobin or HPRT. The choice of reference sample can vary depending on the type of gene expression experiment. For example, a reference sample might be an untreated control sample relative to a treated sample, normal tissue relative to diseased tissue, or liver relative to brain.
For the ΔΔCq method to be valid certain assumptions must be met. First, the reference gene must be stably expressed between different cells of tissues and unaffected by any experimental treatment. Second, the amplification efficiencies of both target gene and reference gene must be approximately equal. Both of the conditions should be experimentally validated for each experiment before the ΔΔCq method can be used.
When extreme accuracy is needed, the Eco software supports use of multiple reference genes as well as PCR efficiency correction.
Illumina recommends using a fluorometric-based method that is specific to double-stranded DNA, such as QuantiFluor or PicoGreen. Consult the DNA Input Recommendations section of the TruSight One Library Prep Guide for additional information.
A fluorometric based method, such as qPCR is recommended for quantifying gDNA.
Illumina recommends quantifying the enriched library using a fluorometric-based method specific to double-stranded DNA, such as QuantiFluor or PicoGreen. For more information, see the Validate Library section of the Nextera Rapid Capture Enrichment Guide.
qPCR can also be used to quantify the final enriched Nextera Rapid Capture Enrichment library. For more information, see the Sequencing Library qPCR Quantification Guide.
Illumina recommends using fluorometric based methods for quantification, including Qubit or PicoGreen to provide accurate quantification of ChIP DNA. UV-spec based methods, such as the Nanodrop, will measure any nucleotides present in the sample including RNA, dsDNA, ssDNA, and free nucleotides which can give an inaccurate measurement of ChIP DNA.
The protocol is optimized for shearing using Covaris fragmentation. Other methods are not supported or tested by Illumina and may result in low yield, unexpected size distributions, or sample failure.
Illumina recommends Covaris shearing, but testing with nebulization shows comparable data.
The protocol is optimized for shearing using Covaris fragmentation. Other methods are not supported or tested by Illumina and may result in low yield, unexpected size distributions, or sample failure.
The MiSeq runs on Windows 7 Embedded Standard.
The ForenSeq DNA Signature Prep Kit is for use with human DNA samples only.
The TruSeq Stranded Total RNA Sample Prep kits with Ribo-Zero Human/Mouse/Rat and TruSeq Stranded Total RNA Sample Prep kits with Ribo-Zero Gold both support human, mouse, and rat organisms. That use of these kits with other organisms has not been tested and is not supported.
Contact your local IT administrator if local security policies have to be modified to allow access to BaseSpace. BaseSpace uses SSL/https port 443 and the domains *.basespace.illumina.com and *.s3.amazonaws.com. Data streaming to BaseSpace is encrypted using the AES256 standard and uses SSL for protection. More information on encryption can be found at http://blog.basespace.illumina.com/2011/12/13/basespace-security/
The instrument computer is a computational engine performing real time analysis of data. To avoid loss of data and other adverse effects, Illumina does not recommend installing any additional software with the exception of anti-virus software.
The ForenSeq DNA Signature Prep Kit includes two positive controls: an assay control, which is processed with the samples through library preparation, and a sequencing control, which is a prepared library included with the pooled samples before sequencing. Two negative controls can also be included: a reagent blank, which is included from the DNA extraction step, and a PCR blank, which is a nuclease-free water sample processed through library preparation.
MiSeq FGx Control Software displays four metrics to use when monitoring the run: Cluster Density, Clusters Passing Filter, Phasing, and Prephasing.
Illumina recommends quantifying your TruSeq ChIP libraries using qPCR according to the Illumina Sequencing Library qPCR Quantification Guide.
The recommended read length for the 350 bp insert size is ≤ 2 X 101 bp and ≤ 2 X 151 bp for the 550 bp insert size. Read lengths greater than those recommended for each insert size will produce a significantly higher percentage of overlapping read-pairs.
A complete list of consumables and equipment required to perform bisulfite sequencing applications can be found in the Whole-Genome Bisulfite Sequencing (WGBS) and Reduced Representation Bisulfite Sequencing (RRBS) protocols. A list of compatible kits can be found on the epigenetics section of the Illumina website .
The run length is specified in the MiSeq FGx software and cannot be changed. The run length is 351 x 31 cycles with 2 x 8 cycle index reads.
For exome or custom enrichment sequencing, TruSeq DNA Sample Prep Kits must be used.
For TruSeq v2/LT kits, standard sequencing primers included in the cluster generation kits are required.
For TruSeq HT kits, primer usage will depend on the flow cell type used. Primers in the TruSeq Dual Index Sequencing primer kit, single read (catalog # FC-121-1003) are needed to run TruSeq HT Dual-Indexed libraries on single read v3 flow cells. These primers are HP10 (read 1), HP12 (Index read 1), HP9 (Index read 2 for single read flow cells only). This add-on box is not required if sequencing a TruSeq HT prepared library with the MiSeq System or on single-read rapid flow cells, or paired-end v3 or Rapid flow cells for HiSeq, HiScanSQ, GAIIx. The primers in the TruSeq Dual Index Sequencing Primer kits are backwards compatible with all Illumina libraries; there is no need to spike in primers as these primers are backwards compatible with other Illumina library types.
TruSeq ChIP libraries are compatible with sequencing primers included in all TruSeq cluster kits as well as in TruSeq Dual Index Sequencing Primer Box, Single Read or Paired End.
Supported read lengths for the HiSeq X are up to 2 x 150 bp plus single 8 bp indexing. Only single-indexed runs are possible on the HiSeq X system.
Dual index sequencing of TruSeq Nano DNA HT libraries on the HiSeq, HiScanSQ, or GAIIx system using single-read flow cells requires the TruSeq Dual Index Sequencing Primer Box, Single Read (FC-121-1003) to be ordered separately. This add-on box is not required if sequencing a TruSeq Nano DNA HT prepared library with the MiSeq System, HiSeq Rapid two-lane single or paired-end flow cells, or on traditional paired-end flow cells for HiSeq, HiScanSQ, GAIIx. Additional SBS reagents may be necessary to account for all index cycles.
If you are not copying files to the target server fast enough, consider the following:
Similar to the Infinium genotyping assay, we recommend DNA that is sized ≥ 2kb, which can be assessed on an agarose gel.
Where .cif files can be generated, you can use OLB 1.9.4.
HiSeq users must upgrade to HCS1.5/RTA1.13 or higher and GA users must upgrade to SCS2.10/RTA1.13 or higher.
Instrument Software and Sequencing Run QC
HCS 2.0/RTA 1.17.20/SAV 1.8.20/Recipe Fragment 1.3.54
SCS 2.10/RTA 1.13/SAV 1.8.20
Sample Sheet Creation
Illumina Experiement Manager 1.3
Please note that SCS 2.10/RTA 1.13 for GA is scheduled to be available November 2011.
The automated processes specific to GoldenGate Assay for Methylation are:
The remaining steps are identical to genotyping using the GoldenGate Assay.
The cDNA strand is sequenced.
After the Illumina FastTrack Microarray Services team receives your signed cont