Questions & Answers

General

  • What is the throughput of the NovaSeq System?

    At launch, the NovaSeq 6000 with the S2 flow cell is capable of generating 2 TB under 2 days. For detailed performance parameters, see the NovaSeq Series of Sequencing Systems Specification Sheet.

Libraries

  • Are custom libraries supported on the NovaSeq System?

    Yes. The NovaSeq Sequencing System permitts custom libraries. However, the NovaSeq denature and dilute protocols might require further titration and optimization to achieve optimal results for custom libraries.

  • What applications can I run on the NovaSeq System?

    The NovaSeq Sequencing System is an open platform suitable for a wide range of methods. These methods include, but are not limited to, whole genome, exome, and transcriptome sequencing. Public data sets are available in BaseSpace Sequence Hub.

Reagents and Flow Cells

  • What kit sizes are available?

    The NovaSeq 5000/6000 S2 Reagent Kit is available in 3 sizes: 300 cycles (2 x 150), 200 cycles (2 x 100), and 100 cycles (2 x 50 or 1 x 100).

  • Are NovaSeq 5000/6000 flow cells available in single-read and paired-end versions?

    NovaSeq 5000/6000 reagent kits include all consumables to support both single-read and paired-end sequencing. During run setup, you can configure the run to generate either single-read or paired-end data.

  • Can I use custom primers on the NovaSeq System?

    Yes, the NovaSeq 5000/6000 Cluster Cartridge and the NovaSeq Control Software allow the use of custom primers for Read 1, Read 2, and the Index 1 Read. Positions 5, 6, and 7 on the cluster cartridge are reserved for custom primers and the control software includes an option to select custom primers during run setup.

  • How often do I need to replace a wash flow cell?

    Replace a wash flow cell when it becomes visibly worn. Wash flow cells do not contain any chemistry, so consistent replacement is not necessary.

  • The flow cell is loose in the cartridge, do I need a replacement?

    No. The flow cell is designed to be loose in the cartridge to ensure proper registration.

  • Which part of the flow cell stage do I clean before loading a flow cell?

    Clean the entire flow cell stage within the flow cell compartment, including the glass surface of the optical alignment target, to remove any visible particulate. The flow cell stage consists of the two flow cell holders and surrounding area.

    Use an alcohol wipe for cleaning, and a lint-free tissue for drying and removing alcohol residue. Cleaning is necessary only if particulate is visible.

     

Workflow

  • What is the maximum read length for a sequencing run?

    The NovaSeq Sequencing System supports read lengths up to 2 x 150 bp.

  • What indexing options are available on the NovaSeq System?

    NovaSeq 5000/6000 reagent kits support single and dual indexing options. The kits include sufficient reagent for 47 additional cycles: 7 chemistry-only cycles plus up to 40 cycles for the index reads. The indexing primer provides primers for both single and dual indexing. The NovaSeq System follows the same dual indexing workflow as the HiSeq 2500 system.

  • What reagents are required for an instrument wash?

    The automatic post-run wash uses diluted sodium hypochlorite (NaOCl) included in the cluster cartridge. The maintenance wash, which is required every 14 days the instrument is idle, requires user-supplied dilutions of Tween 20 and NaOCl.

Software

  • Can I use Sequencing Analysis Viewer to view data from a NovaSeq run?

    Yes. Sequencing Analysis Viewer (SAV) v1.11 or later is required to view data from a NovaSeq System run. You can download SAV for use on a computer or use BaseSpace Sequence Hub.

  • How much space is needed on the output drive?

    Each cycle of a run requires at least 5.4 GB of space. Therefore, a typical 2 x 150 bp mid-output run requires about 1.7 TB of space. For long-term storage, save nonsequencing data to external storage instead of the instrument. Use the Disk Management screen to view available disk space, transfer the run data to external storage, and clear disk space for a new run.

Analysis

  • What are my options for analyzing NovaSeq sequencing data?

    BaseSpace Sequence Hub is the preferred analysis solution for the NovaSeq Sequencing System. On-instrument analysis is not available. For third-party and user-developed analysis packages, you can use the bcl2fastq2 converter.

  • What version of bcl2fastq is supported for the NovaSeq System?

    bcl2fastq v2.19 is supported for processing NovaSeq data.

  • If a network outage to the central file server storing a runs occurs, can NovaSeq cache the entire 2 x 150 run?

    Yes. The server can store as many runs as disk space permits. Real-Time Analysis continues processing and resumes data transfer when the network is restored.

  • What are CBCL files?

    CBCL files (*.cbcl) are concatenated BCL files that save space and optimize output. Instead of generating BCL files on a per tile basis, tiles from the same lane and surface are aggregated into 1 CBCL file for each lane and surface.

  • How does 3+1 quality binning work, and how does it compare to 7+1 without binning?

    The 3+1 quality binning permits a reduction in the data file sizes generated by the NovaSeq System. The mapped Q-scores for each bin are Q12, Q23, and Q37. Testing has shown highly comparable performance in HWGS between NovaSeq 3+1 qualities and HiSeq X 7+1 qualities. Although third-party analysis solutions have shown comparable performance, Illumina recommends recalibrating any settings used.