MiSeq FAQs

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  • Libraries


  • Yes, you can sequence your enriched libraries on the MiSeq System, obtaining results in hours instead of days. However, MiSeq Reporter currently cannot process the manifest file provided for your enrichment experiment. Therefore, 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 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 generates the standard Resequencing reports. Zoom in on your enriched regions to obtain relevant data.

    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.

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

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

    Visit the MiSeq Products page for a comprehensive list of fast, easy library prep options. To determine which option is best suited for your application, see Sample Prep Applications for the MiSeq System.

    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 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.

    The MiSeq System 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.

    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.

    The HiSeq v4 reagent kits 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.

  • Workflow


  • 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 v2 reagents.

    All MiSeq System runs include an automated wash, which rinses the primary fluidics tube without requiring user intervention.

    However, the automated wash does not replace the post-run wash, which must be performed after every run to flush any remaining reagents from the fluidics tubes and sippers and prevent salt accumulation, crystallization, and cross-contamination from the previous run.

    Perform a maintenance wash every 30 days.

    The MiSeq manifest folder contains the manifest files required for custom amplicon and PCR amplicon workflows. A manifest file is required to specify alignments to a reference and the targeted reference regions.

    You can specify the location of the manifest folder using the MiSeq control software interface. Before starting the run, copy the manifest file to the specified location.

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

    • The TruSeq controls were not designed to be a qualitative metric of the efficiency of the various steps in the library 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.

    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 Guide.

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

    You can perform up to 2 x 250 bp, or 500 cycles of sequencing, on the MiSeq using the MiSeq Reagent Kit v2. However, some applications tolerate as few as 36 cycles.

    The MiSeq Reagent Kit v3 allows up to 2 x 300 bp, or 600 cycles of sequencing. The MiSeq Reagent Kit v3 is available in two sizes: 600-cycle and 150-cycle.

    Dual-indexed runs on HiSeq systems comprise 8 bp of index sequence rather than 6 bp plus a seventh for phasing calculations. For more information, see the system guide for your sequencing system.

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

    With MiSeq Control Software (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.

    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.

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

    The post-run wash takes approximately 30 minutes.