Questions & Answers

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General

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 Specification Sheet.

The NovaSeq Xp workflow enables sequencing of different libraries (pools) in each lane of the NovaSeq flow cell. The ExAmp/library mix is prepared manually and loaded onto the flow cell using the NovaSeq Xp Flow Cell Dock. 

No. Run times are the same between the Standard workflow and the NovaSeq Xp workflow.

Libraries

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.

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.

The optimal library loading concentrations for the NovaSeq Xp workflow is lower than for the Standard workflow.  Refer to the NovaSeq System Guide for library concentration in the final ExAmp/library mix. 

Output is delivered by lane. Hence, plex level of the library pool needs to be reduced accordingly. 

Applications supported by the Standard workflow are also supported by the NovaSeq Xp workflow. Refer to the NovaSeq Specification Sheet.

Yes. Different library pools separated by projects or by sequencing methods can be loaded in each lane of the NovaSeq flow cell using the NovaSeq Xp workflow. 

Depending on the optimal loading concentration, savings in DNA is at least 60%. 

Reagents and Flow Cells

The NovaSeq 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).

NovaSeq 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.

Yes, the NovaSeq 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.

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

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

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.

 

The NovaSeq Xp kit is available in two sizes. The NovaSeq Xp 2-lane kit is used with S1 and S2 flow cells. The NovaSeq Xp 4-lane kit is used with S4 flow cells. Each kit contains a single use manifold and sufficient reagents for one NovaSeq flow cell.

Yes, the reagents can go through one additional freeze/thaw cycle. However, left over reagents should not be refrozen. 

Manual ExAmp Step

  1. Make sure to use the correct DPX volumes for your flow cell type.
  2. Dispense slowly enough to make sure that entire volume is expelled from the pipette tip.
  3. Prepare fresh ExAmp master mix. It works best when used immediately.
  4. The most common cause of variability in data quality when manually mixing ExAmp are inaccurate delivery of ExAmp component volumes and insufficient mixing. Make sure to vortex mixes thoroughly. 

Freshly mixed ExAmp master mix works best. If immediate use is not possible, ExAmp mix kept on ice must be used within an hour, or within 30 min when stored at room temperature. Ideally, ExAmp mix is kept on ice prior to library addition. 

Yes. Vortexing is the recommended mixing method for the NovaSeq Xp workflow. 

No, we do not recommend combining leftover reagents from multiple kits. 

Lane Filling

We have not observed cross-contamination between lanes. Following good lab practices, such as keeping the loading area and the flow cell area on the instrument clean and avoiding dripping the ExAmp/library mix over the flow cell when removing the manifold, is important. 

No. Small bubbles are normal and have no impact on sequencing quality. They appear in 80% of fill events and have no impact on sequencing quality. 

No. Staging a loaded flow cell at any temperature is not recommended. It is best to complete the NovaSeq Xp workflow and proceed to sequencing immediately. 

The sequencing run should be initiated within 30 minutes after loading the ExAmp/library mix onto the flow cell. Depending on library, extended staging at room temperature has the potential to result in low %PF. PCR-free libraries demonstrate a greater drop in % PF upon extended staging. 

Leaving lanes empty is not recommended. Lanes can be left empty depending on the type of flow cell being run. For 2-lane flow cells, an empty flow cell lane will result in certain auto-center failure and the run will not start. For 4-lane flow cells, no more than one lane can be left empty for successful auto-center. 

Call technical support for further guidance. Generally, partial fill in one lane of a 2-lane flow cell (S1 or S2 flow cells) will result in auto-center failure. However, partial fill in one lane of 4-lane flow cell (S4 flow cell) can be successfully sequenced to completion.

Workflow

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

NovaSeq 6000 reagent kits support single and dual indexing options. The kits include sufficient reagent for 25 additional cycles to support 7 chemistry-only cycles plus up to 16 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.

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

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.

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

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.

bcl2fastq v2.19 is supported for processing NovaSeq data.

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

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.

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.