MiSeq FAQs

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 the  MiSeq System Custom Primers Guide (document # 15041638).

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.

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

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

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

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.

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 System performs cluster generation followed by sequencing and paired-end chemistry (if applicable).

No. The MiSeq flow cell is specifically designed for the MiSeq System.

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

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, a primer rehyb is not possible on the MiSeq System.

MiSeq Reagent Kits v3 are kitted for 150 cycles and 600 cycles.

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

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.

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.

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

Yes. There are two new reagents in MiSeq Reagent Kit v3: new Incorporation Mix (IMT) and new Scan Mix (USM), and the PR2 volume is increased to 500 ml to support longer runs.

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.

This step takes approximately 70 minutes.

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.

You can install a second licensed copy of MiSeq Reporter Software on another computer. However, that computer must be connected to the same network as your MiSeq System 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.

The MiSeq System runs on Windows 7 Embedded Standard.

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

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.

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

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 Software Guide.

No. MiSeq Reporter is used to view MiSeq data. For an overview of the software, see the MiSeq Reporter page.

Depending on cluster density, metrics appear at the beginning of cycle 20. For MCS v2.2 or earlier versions, metrics appear at the beginning of cycle 7.

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.

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.

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.

Set up your sample sheet to use the GenerateFASTQ workflow, which generates FASTQ files without proceeding to further analysis. FASTQ files can be exported for analysis with third-party software.

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

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.