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.