Questions & Answers

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Primers

Can custom primers be used on the MiSeq

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).

Libraries

  • Can I sequence TruSeq Custom Enrichment libraries on the MiSeq?

    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:

    • Select Workflow: select MiSeq Reporter and the Resequencing workflow.
    • Select Compatible Assay: select TruSeq DNA/RNA.

    MiSeq Reporter will generate the standard Resequencing reports. Consequently, you will need to zoom in on your enriched regions to obtain relevant data.

  • How do I perform amplicon sequencing on a MiSeq?

    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.

  • Can I run libraries prepared for the HiSeq or Genome Analyzer on the MiSeq?

    Yes. You can run any library on the MiSeq provided your library has complementary adapters.

  • Which applications are supported on the MiSeq?

    MiSeq Reporter, the secondary analysis software on the MiSeq, supports amplicon sequencing, targeted resequencing, small genome sequencing (de novo/resequencing), and small RNA applications. For complete information, see Sample Prep Applications for the MiSeq System.

  • What sample prep do I use for MiSeq?

    A comprehensive list of easy and rapid sample prep options can be found on the MiSeq kits page. To determine which sample method is best suited for your application, see Sample Prep Applications for the MiSeq System.

  • What controls should I use for sequencing low-diversity samples on a MiSeq?

    Illumina recommends adding PhiX at 1% to all libraries as an internal control. For low-diversity libraries, the percentage of PhiX depends on the diversity of the library and requires optimization. Monotemplates may require at least 30% PhiX. If you are using RTA 1.17.28 (released with the MiSeq v2.2 updater) or later, low-diversity libraries requires a minimum of 5% PhiX spike-in. A PhiX control is not required for libraries prepared with the TruSeq Custom Amplicon Kit or the Nextera XT kit.

  • How much template does the MiSeq use?

    The MiSeq uses a total of 500 µl of denatured and diluted template for priming and clustering. However, 600 µl of template is recommended to avoid air bubbles and accommodate for instrument sipper depth. This volume does not vary for different workflows.

  • What is the minimum PhiX amount that can be spiked in?

    For most libraries, Illumina recommends using a low-concentration spike-in (1%) of PhiX Control v3 as an in-lane positive control for alignment calculations and quantification efficiency.

  • Can I use only 1 of the indexes of a dual-indexed library?

    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.

Reagents and Flow Cells

  • What cluster generation and sequencing reagents do I use for MiSeq?

    The MiSeq System uses the MiSeq Reagent Kit, which includes specially designed and packaged reagents for cluster generation and sequencing.

  • How many lanes does the MiSeq flow cell have?

    The MiSeq flow cell is a single-lane flow cell.

  • Are the RFID tags for the flow cell and reagent cartridge linked in any way?

    No. The flow cell RFID is unique to the flow cell and reagent cartridge RFID is unique to the reagent cartridge.

  • Which reagent position is used for the MiSeq flow check?

    During the pre-run flow check, reagent is pulled from position 3 on the reagent cartridge, which is PR2.

  • How many cycles can I perform with the MiSeq Reagent Kit?

    Illumina offers a variety of kit sizes ranging from a 50-cycle kit to a 600-cycle kit. MiSeq Reagent Kit v3 is available in sizes of 150 cycles and 600 cycles. The MiSeq Reagent Kit v2 is available in sizes of 50 cycles, 300 cycles, and 500 cycles. For more information, see the MiSeq Reagent Kit support page.

  • How is flow cell clustering conducted on MiSeq?

    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).

  • Are other sequencing flow cells compatible with MiSeq?

    No. The MiSeq uses a flow cell that is specifically designed for the MiSeq.

  • How many empty ports are available on the reagent cartridge?

    Three reservoirs on the MiSeq reagent cartridge are available for user-supplied custom primers. For more information, see Using Custom Primers on the MiSeq (part # 15041638).

  • Can I use HiSeq or Genome Analyzer reagents on the MiSeq?

    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.

  • Can I perform a primer rehyb on the MiSeq?

    No, a primer rehyb is not possible on the MiSeq.

  • How many reads can I expect from an optimally clustered MiSeq flow cell?

    You can expect about 15 million reads passing filter.

  • Does the MiSeq reagent cartridge contain sequencing primers for all applications?

    Yes. The MiSeq reagent cartridge contains sequencing primers for TruSeq DNA and RNA samples, TruSeq Small RNA samples, TruSeq Custom Amplicon libraries, and Nextera DNA samples.

  • How many cycles do MiSeq v3 kits support?

    MiSeq v3 kits are kitted for 150 cycles and 600 cycles.

  • Can I thaw the reagent cartridge in the refrigerator overnight?

    For best results, thaw the reagent cartridge in a room temperature water bath for approximately 60 minutes.

  • Are flow cells provided in MiSeq v3 kits different from flow cells provided in MiSeq v2 kits?

    No. The flow cells are identical. However, the upgrade to MCS v2.3 enables imagine of the flow cell in 19 tiles per surface. MCS v2.3 is required software for the MiSeq Reagent Kit v3.

  • What do I need to run MiSeq v3 chemistry?

    Using reagents provided in the MiSeq Reagent Kit v3 requires the recipes and RFID recognition settings in MiSeq Control Software (MCS) v2.3. To download a copy of MCS v2.0, see the MiSeq System downloads page.

  • What do I need to run MiSeq v2 chemistry?

    Using reagents provided in the MiSeq Reagent Kit v2 requires the recipes and RFID recognition settings in MiSeq Control Software (MCS) v2.0. To download a copy of MCS v2.0, see the MiSeq System downloads page.

  • What is the shelf life of the MiSeq Reagent Kit?

    Illumina guarantees that all reagent products will ship with a minimum viable shelf-life of three months.

  • Are there changes to reagents in the MiSeq v3 kits?

    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.

Workflow

  • Is there a 300-cycle MiSeq v3 reagent kit?

    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.

  • What washes are recommended for the MiSeq?

    All MiSeq runs include an automated wash, which rinses out the primary fluidics line, and does not require user intervention. However, the automated wash does not replace the need for a post-run wash, which should be performed after every run to flush any remaining reagents from the fluidics lines and sippers, and prevent salt accumulation, crystallization, and cross-contamination from the previous run. A maintenance wash should be performed every 30 days.

  • What is a MiSeq manifest folder?

    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.

  • Can I run a control lane on a MiSeq flow cell?

    No, a dedicated control lane is not possible because the MiSeq flow cell a single-lane flow cell. Illumina recommends using a spike-in method for adding a control to libraries.

  • How do I know if the TruSeq controls have worked?

    • The TruSeq controls were not designed to be a qualitative metric of the efficiency of the various steps in the sample prep but rather an indicator of whether or not the step worked or not. Actual counts of the controls will vary based on sample type, input, etc.
    • In SAV (Sequencing Analysis Viewer), if you see counts for a particular control, this mean that this step has worked whereas if you see no counts (dark blue color), this step has likely failed. If the band has been cut across multiple insert sizes, you may see counts at different size increments.

  • Should I leave my MiSeq on when not in use?

    It is best to leave the instrument on at all times. However, if you need to turn off the instrument, perform a maintenance wash first, and then use the Shut Down command on the Manage Instrument screen to safely shut down Windows. If the instrument will not be used within seven days, make sure that you prepare the fluidics system to sit idle. For more information, see the MiSeq System User Guide, Part # 15027617.

  • What if I did not name the sample sheet with the reagent cartridge barcode number?

    During run setup, the software searches for a sample sheet of a name that matches the barcode number of the reagent cartridge scanned in the previous run setup step. If a sample sheet of this name is not found, the software will pause and prompt you to browse to the apppropriate sample sheet for this run, regardless of the sample sheet file name. Naming the sample sheet with the reagent barcode number skips this additional step.

  • What is the read length on MiSeq?

    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.

  • How long does it take to complete a 600-cycle run on the MiSeq?

    A 600-cycle run takes approximately 55 hours.

  • For dual-indexed libraries, how many cycles are performed for index reads?

    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.

  • How long does it take to perform one full cycle on the MiSeq?

    One full cycle takes approximately 6 minutes and consists of a chemistry cycle and an imaging cycle.

  • How long does template generation take on the MiSeq?

    With MCS v2.3, template generation occurs during the first 7 cycles of sequencing. When using earlier versions of MCS, template generation occurs during the first 4 cycles of imaging.

  • How long does it take to perform cluster generation and primer hybridization on the MiSeq?

    This step takes approximately 70 minutes.

  • How long does it take to complete a 150-cycle run on the MiSeq?

    A 150-cycle run takes approximately 20 hours.

  • How long does it take to complete a run on the MiSeq?

    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.

  • Do I need to use a cBot with the MiSeq?

    No. Cluster generation is performed on the MiSeq instrument as part of the run. No other instruments are required for sequencing on the MiSeq.

  • How long does it take to perform a post-run wash on the MiSeq?

    The post-run wash takes approximately 30 minutes.

Software

  • Can I run MiSeq Reporter on a computer independent of the MiSeq?

    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.

  • What operating system is used on the MiSeq?

    The MiSeq runs on Windows 7 Embedded Standard.

  • I am currently finishing a project using MiSeq v2 reagents. Can I upgrade my software to MCS v2.3 and still finish my project with v2 reagents?

    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.

Analysis

  • Can GenomeStudio be used to view MiSeq data?

    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.

  • Can Sequencing Analysis Viewer (SAV) be used with the MiSeq to view primary analysis results?

    Yes. You can install SAV on another computer that is connected to the same network as the instrument. MiSeq data will appear when you select All or Lane 1 from the drop-down menu. For more information, see the Sequencing Analysis Viewer User Guide.

  • What is the error rate on MiSeq?

    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.

  • What if I want to do an experiment that does not fit into one of the MiSeq Reporter analysis workflows?

    You can set up your sample sheet to use the GenerateFASTQ workflow, which generates FASTQ files and does not proceed to further analysis. FASTQ files can be exported for analysis by a third-party software.

  • Can the MiSeq generate FASTQ files?

    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.

  • Where is the status.htm file?

    This report is not created by the MiSeq. However, similar information can be viewed using Sequencing Analysis Viewer (SAV).

  • How do I analyze data from a run on the MiSeq?

    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.

  • Can I expect changes when using Sequence Analysis Viewer (SAV) with MCS v2.3 and MiSeq v3 reagents?

    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.

  • How do I deselect MiSeq Reporter when I start a run?

    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.

  • Can CASAVA be used to analyze MiSeq data offline?

    Yes, CASAVA can be used to analyze MiSeq data offline.  The MiSeq output folders are readable by CASAVA without a need to modify any configuration files.

  • When will quality scores appear during a sequencing run on the MiSeq?

    Quality scores appear after cycle 25. The first 25 cycles are used to determine the chastity of a base call, which in turn determines the quality filter.

  • How long does it take from the start of the run until I have cluster density metrics?

    Depending on cluster density, metrics appear at the beginning of cycle 20, if you are running MCS v2.3. With earlier version of MCS, metrics appear at the beginning of cycle 7.

  • What criteria determine clusters passing filter on Illumina sequencing systems?

    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.

Can secondary analysis run on MiSeq while a run is in progress?

If a new sequencing run is started on the MiSeq before secondary analysis of a previous run is complete, secondary analysis will be stopped automatically. MiSeq computing resources are dedicated to either sequencing or analysis, and the system is designed in such a way that a sequencing command overrides an analysis command. Secondary analysis can later be requeued from the MiSeq Reporter Analyses tab.