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.
Yes. Single read and paired end libraries for RNA and DNA applications are compatible with the HiSeq.
Sample prep for dual-indexed libraries requires that both indices 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 eight-base single-indexed sequencing run.
Homopolymers do not impact sequencing. The number of uniquely alignable reads is a function of the repeat content, so this will have an impact on productivity. With longer reads and paired-end sequencing, this may be less of an issue.
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.
No. The HiSeq flow cell, also used on the HiScanSQ, must be clustered on a cBot equipped with an adapter plate suitable for the larger format of the flow cell.
Due to an interaction with one of the v3 reagents, SRE, waste appears dark brown in color and has a stronger odor. This is normal. The change in waste color does not impact performance and is not toxic. You might see a discoloration on the funnel caps and SRE sipper line. Any spills will be dark brown in color as well.
Yes. You can split the 200-cycle SBS Kit into two equal volumes suitable for up to 101 cycles each. See the TruSeq SBS Kit Reagent Preparation Guide (200 Cycles) for instructions and storage requirements. If you need smaller volumes for shorter runs, Illumina recommends using the TruSeq SBS Kit (50 Cycles).
No. The HiSeq and HiScanSQ do not require immersion oil to properly load a flow cell in the way that the Genome Analyzer does. Instead, the flow cell is held in place by a vacuum, which removes air and replaces the need for immersion oil.
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. The TruSeq SBS Kit (200 Cycles) contains sufficient reagents for 209 cycles of sequencing, which covers 101 cycles for Read 1, 7 cycles for the Index Read, and 101 cycles for Read 2.
Illumina currently offers two SBS kits for the HiSeq: a 200-cycle kit and a 50-cycle kit. Both kits contain the same formulations and only differ in volume.
No. Cluster Kits and SBS Kits for the HiSeq are not equivalent or compatible with the Genome Analyzer. Likewise, Cluster Kits and SBS Kits for the Genome Analyzer are not equivalent or compatible with the HiSeq.
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.
Yes. The HiSeq flow cell, also used on the HiScanSQ, requires the use of a cBot for clustering on the flow cell prior to sequencing.
Flow cells are designed for single-use. All eight lanes must be used at the same time. They can be used for the same sample or for different samples. You can run eight samples at a time without multiplexing. With multiplexing, you can increase throughput to up to 12 samples per lane or up to 96 samples per flow cell.
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.
When using v3 SBS reagents and the v3 flow cell on HiSeq, each additional index cycle is approximately 53 minutes per cycle (with 16 total indexing cycles) plus ~2 hours for the seven chemistry-only cycles for the PE workflow, resulting in ~16 hours of additional time for dual indexing on HiSeq.
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.
With HCS v1.3 and later, you can customize a recipe to contain any number of reads. Reads can be indexed or non-indexed. However, Illumina does not guarantee the performance of custom recipes. Contact your Illumina Technical Support if you need assistance creating custom recipes.
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:
The HiSeq 2000 is a dual flow cell system, which allows you to run two flow cells simultaneously. The HiSeq 1000 is a single flow cell system.
No, the system does not support an initial cycle indexing method. To ensure the highest quality data, Illumina recommends and supports a separate indexing read for multiplexed samples.
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.
No. You can choose to run only one flow cell at a time.
No. You can start each flow cell independently from the other. Each flow cell can have a different number of reads and cycles.
Dual-indexed runs on the HiSeq comprise 8 bp of index sequence rather than 6 bp plus a seventh for phasing calculations.
Please 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.
Index reads for single-read libraries use seven cycles reads. Illumina does not support six cycle index reads for single-indexed libraries.
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 sample prep in order to confirm appropriate index combinations.
Yes. Illumina recommends using a PhiX control lane when sequencing ChIP-Seq libraries. Samples that contain genomes with high AT or GC content (less than 40% or greater than 60%) require a dedicated PhiX control lane for cross-talk and phasing calculations. For more information, see Using a PhiX Control for HiSeq Sequencing Runs.
A PhiX spike-in employs a small amount of PhiX control in the same lane as a sample. This allows real time quality metrics as the PhiX is analyzed during the run. This is not recommended for sequencing a genome with high similarity to the PhiX genome, and does not allow for normalization of data in that lane as per a control lane.
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.
This error message indicates a lack of fluorescence on the flow cell. To find focus at the start of a run, the software uses ETF, which is a focusing method that reads fluorescence from clusters on the flow cell. ETF must find fluorescence in at least one lane of the flow cell before the run can begin.
To correct this problem, perform a primer rehybridization. Re-annealing the Read 1 sequencing primer usually increases the fluorescence if clusters are present on the flow cell. Additionally, check the cBot plate to make sure that all reagents were delivered correctly and that the sequencing primer was appropriate for your library types. When you reload the flow cell on the HiSeq, confirm that the fluidics system is functioning correctly. If a primer rehybridization does not resolve the issue, 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.
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.
No. Images are deleted automatically after they have been processed.
Yes. The *.cif files can be saved and transferred to the analysis server over the network.
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.
Images are taken using a time delayed integration (TDI) line scanning optical system with four CCD sensors. The TDI line scanning system greatly increases throughput by maximizing camera utilization.
HCS allows you to designate a control lane during the run setup steps. Generally, you do not need to designate a control lane if the sequence you are analyzing has a balanced genome. In the case of an unbalanced or skewed base composition (e.g., bisulfite-treated samples) a control lane is recommended. This is not equivalent to a PhiX spike-in.
The HiSeq instrument computer employs 64-bit Windows Vista.
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 first time the HCS 2.x (or later) is launched, you will see a notification regarding instrument health data. This notification will appear 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. If the customer is not present to view this notification, Illumina staff will refer the customer to the Options menu and ensure that they are aware of this setting.
The following files are uploaded as instrument health data: RunInfo.xml, RunParameters.xml, RTAComplete.txt, InterOp files, and RTAConfiguration.xml.
HCS software allows HiSeq instruments connected to the internet to send instrument health information to Illumina. This information will be anonymous and will include only generic run metrics. This information will be used by Illumina to help improve Illumina’s products. If you wish to turn off this option, or would like further information, please go to the Options menu in the HiSeq control software. You can find the Options menu under Menu|Tools.
The BaseSpace run monitoring option enables you to monitor an ongoing run’s progress remotely by logging into your BaseSpace account. You will need to select the Run Monitoring option during run setup. Then, you can log into your BaseSpace account from anywhere and view your run in the BaseSpace version of Sequence Analysis Viewer.
The HiSeq will send only the InterOp folder, RunInfo.xml file, and RunParameters.xml file.
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.
No. Thumbnail images are for visual inspection only to help diagnose problems with a run. They are not suitable for reanalysis.
Storage requirements for raw data are approximately 60% greater than current runs based on additional swath data and increased cluster density.
You need a one gigabit connection per instrument between the instrument computer and the server. For more information, see the HiSeq System Site Preparation Guide.
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.
Yes. HCS 2.x enables you to save cifs.
If a run has zipped bcls, you will need to use a new software tool, Bcl2Fastq, 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 zipped or non-zipped bcl files.
If you send your data to BaseSpace, bcl to fastq conversion and demultiplexing will be performed automatically following the completion of the data upload.
If you want to upload data from a HiSeq to BaseSpace, you will need a minimum upstream connection bandwidth of 10Mbit/second per instrument. You can assess network speed using free online tools, such as www.speedtest.net.
Please see the Illumina whitepaper, Reducing Whole-Genome Data Storage Footprint, which is available on MyIllumina. You can find it on the HiSeq Documentation and Literature support pages.
HCS 2.x features the two data compression options: bcl file zipping and Qscores binning. These options are available during run setup in HCS 2.x. If you are using BaseSpace for data storage and analysis, bcl files will be zipped by default. If you are using BaseSpace for only run monitoring, bcl zipping is not required. Other run folder files are unchanged from previous versions of HCS.
If you are not using BaseSpace, the data compression options are as follows:
1. No compression
2. Zip bcls + bin Qscores
3. Zip bcls only
If you are using BaseSpace, the data compression options are as follows:
1. Zip bcls only (required for BaseSpace)
2. Zip bcls + bin Qscores
Scanning and analysis of a Rapid two-lane flow cell creates two swaths per surface on two surfaces per lane. Each swath is divided into 16 tiles; for a two lane flow cell there are a total of 128 tiles per flow cell.
Image analysis occurs in real time, phasing estimates and base calling begin occur after cycle 12, and base call quality scoring occurs after cycle 25.
It is the ability to distinguish between two or more clusters that are in close proximity to each other.
To remove the least reliable data from the analysis results, often derived from overlapping clusters, raw data is 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 BaseSpace Broker is designed to upload data to BaseSpace as soon as the data is 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 the majority of cases, throttling of the BaseSpace Broker data upload should not be necessary. Throttling may be needed if you need to apply greater control over network bandwidth usage (such as at sites at which instruments share the network with other users or sites with limited upload speed). Throttling may be necessary in certain scenarios, such as when local network connectivity is temporarily lost and then restored, causing BaseSpace Broker to suddenly consume more network bandwidth as it tries to “catch up” with the upload of accumulated data. In these types of scenarios, if no throttling is applied, BaseSpace Broker could consume all available bandwidth on the network until the backlog of data is cleared. If throttling is to be applied and if the local network permits, Illumina suggests throttling to higher than the 10Mbit/sec minimum specification. 20Mbit/s (approximately 3Mbyte/s = 24Mbit/s) is recommended. This will provide BaseSpace Broker with enough bandwidth to recover in the case of data upload delays.
If you need to throttle BaseSpace Broker, please supply the following instructions to your local IT administrator:
Throttling of BaseSpace is done on the HiSeq-PC by application rather than by IP address.
1. In windows, open the Local Policy Editor by opening a cmd window and running gpedit.msc.
2. Expand the Computer Configuration / Windows Settings nodes.
3. Select Policy-based QoS.
4. Right-click and then choose Create new policy. Then, perform the following steps:
a. Enter a name.
b. Uncheck Specify DSCP value.
c. Select Specify Outbound Throttle Rate, and then enter 3 Mbytes/s (or 24Mbit/s). This should enable the system to catch-up if necessary.
d. Select Next.
e. Select Only applications with this executable name, and then enter Illumina.BaseSpace.Broker.exe.
f. Click Next (this policy applies to any source IP and target IP addresses).
g. Click Finish (this policy applies to all ports and protocols).
You can use OLB 1.9.4. However, please note that OLB 1.9.4 will be replaced by RTAOLB, which is a means of running RTA in a Linux environment. An RPM of RTA plus needed libraries and instructions will be released on My Illumina in November of 2012.
With Casava, you can merge data from different HiSeq flow cells (different runs) with Casava 1.8.2’s configureBuild script.
First, align the data (samples) from each flow cell with Casava 1.8.2 separately, using configureAlignment. Then, include each Sample Directory as an input directory in the configureBuild.pl command line. You can specify input directories by using the –id option, as detailed on page 100 of the Casava 1.8.2 User Guide Rev C.
With BaseSpace, a sample merge application is available, which enables you to merge data for a single sample that originated from different flow cells. Merging is performed prior to alignment analysis of the sample data.
bcl2fastq is a separate piece of stand-alone software that is run on a Linux scientific computing system. You can get the installer from MyIllumina. Dependencies are equivalent to those for Casava 1.8.2 and are outlined in the bcl2fastq user guide. They are also listed on pages 114 to 117 of the Casava 1.8.2 User Guide Rev C. If the option to compress bcl files was selected during run setup, then conversion of bcl files will require the use of the bcl2fastq.
Yes. bcl2fastq is used in place of the configureBcl2fastq step in Casava. The output of bcl2fastq is fastq.gz files organized into Project and sample directories as specified in the sample sheet. This output will be compatible with the configureAlignment and configureBuild components of Casava 1.8.2. The sample sheet format required for bcl2fastq is equivalent to Casava 1.8.2 sample sheet format, and it is described in the Bcl Conversion with bcl2fastq User Guide.
You will need bcl2fastq to handle zipped bcl files. It can also handle regular (non zipped) bcl files.
You must select run monitoring on the Storage tab of HCS during Run Configuration.
If you choose to use BaseSpace for Run Monitoring only, a sample sheet is not required. If you want to use BaseSpace for data storage and analysis, a sample sheet is required. It must be in Casava format, and all samples listed in BaseSpace must be indexed or all must not be indexed (for example, you cannot have a mix of indexed and unindexed samples in a single flow cell when using BaseSpace).
HiSeq Run data can be uploaded to BaseSpace only during a sequencing run and only if you have selected the BaseSpace option in HCS. Please see the HiSeq User Guide for information on setting up a run for upload to BaseSpace.
For more information on BaseSpace, or to set up a free BaseSpace account, please go to https://basespace.illumina.com/home/index.
No testing has been done on the effects of local proxies on BaseSpace access.
Please contact your local IT administrator if local security policies will need to be modified to allow access to BaseSpace. BaseSpace uses SSL/https port 443 and the domains api.basespace.illumina.com and basespace.illumina.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/
No. Even if you do choose to save cifs, they will be saved only locally and will not be transferred to BaseSpace.