HTG NovaSeq 6000 Sequencing

What types of flow cells are available for the NovaSeq 6000?

Four types of flow cells are currently available for sequencing on the NovaSeq platform: the S4 flow cell (four lanes per flow cell), the S2 flow cell (two lanes per flow cell), the S1 flow cell (two lanes per flow cell), and the SP flow cell (two lanes per flow cell).

What is the average number of read-pairs per lane on a NovaSeq 6000 flow cell?

• Illumina documents the S4 flow cell to deliver 2.0 to 2.5 billion read-pairs per lane.
• Illumina documents the S2 flow cell to deliver 1.6 to 2.0 billion read-pairs per lane.
• Illumina documents the S1 flow cell to deliver 600­–800 million read-pairs per lane.
• Illumina documents the SP flow cell to deliver 300–400 million read-pairs per lane.

What are the standard sequence read lengths supported on the NovaSeq 6000 at the High Throughput Genomics Shared Resource?

Sequence runs with read lengths of either 50x50 bp or 150x150 bp are routinely performed on the NovaSeq 6000 at the High Throughput Genomics Shared Resource. Other read length options including 100x100 and 250x250 bp are also available in addition to custom read length configurations.

What is the maximal read length for a sequence run on the NovaSeq 6000?

The NovaSeq 6000 can support read lengths up to 250x250 bp on an SP flow cell. Other classes of flow cells (S1, S2, and S4) support read lengths up to 150x150 bp.

What sequencing options are available on the NovaSeq 6000 when the High Throughput Genomics Shared Resource constructs the libraries?

A researcher can order sequencing data by either (a) 100 million read-pair increments, (b) full lanes, or (c) full flow cells. Full lanes or flow cells may add cost savings for larger projects. Standard read lengths performed at the High Throughput Genomics Shared Resource include 50x50 bp and 150x150 bp sequence runs. Alternative run lengths and custom read length options can be supported when the customer orders a full flow cell.

What sequencing options are available on the NovaSeq 6000 for libraries constructed by the customer?

A customer that constructs their own libraries can order sequencing data by either (a) full lanes or (b) full flow cells. Standard run lengths include 50x50 bp and 150x150 bp sequence runs. Alternative run lengths and custom read length options can be supported when the customer orders a full flow cell.

What is the library loading molarity requirements for a sequence run on the NovaSeq 6000?

A volume of 18 µl (SP flow cell lane) to 30 µl (S4 flow cell lane) of a 1.0 to 1.5 nM library is required for sequencing on a NovaSeq 6000. Quality control analysis (Qubit assay, Agilent TapeStation assay and qPCR) require additional volume.

How many libraries can be pooled and sequenced in a lane on the NovaSeq 6000?

Library preparation kits used by the High Throughput Genomics Shared Resource include support for 96 to 384 unique dual indexes. Although hundreds of libraries can be sequenced in a single lane on the NovaSeq 6000, each lane yields a finite quantity of data. Therefore, as more libraries are loaded in a lane, the quantity of data assigned to each individual library will be decreased.

Should I be concerned if adapter dimer products are present in my sequencing library?

Patterned flow cells used on the NovaSeq platform tend to be more sensitive to adapter dimer representation in sequencing libraries. A library that contains 5% adapter dimer by molar fraction may be expected to yield as much as 65% adapter-only sequence reads. It is important for researchers that construct their own sequencing libraries to select against the presence of adapter dimers during library purification.

How do I know if my library contains adapter dimers?

Adapter dimers exhibit a size distribution of approximately 130 to 140 bp in libraries that include dual indexed adapters.

What is the optimal insert size for libraries that are sequenced on the NovaSeq 6000?

The NovaSeq flow cell is designed around a defined pattern of billions of nanowells located at fixed positions to enable maximal cluster density, even spacing between clusters, and uniform size distribution. Due to the tight arrangement of nanowells in a sequencing lane, the NovaSeq flow cell works best with libraries containing an insert size that ranges between 50 and 700 bp. Adapter sequences will add an additional 130 to 140 bp to the library.

How does the High Throughput Genomics Shared Resource qualify libraries prior to sequencing?

Quality control assays are performed to validate libraries prior to sequence analysis on the NovaSeq 6000. These assays include the following: Qubit dsDNA High Sensitivity Assay (library concentration), Agilent ScreenTape Assay (size distribution), and qPCR with the KAPA Library Quantification Kit for Illumina Platforms (normalize library representation in preparation for pooling). The cost for these quality control assays is included as part of the library preparation process when the High Throughput Genomics Shared Resource constructs the library. Alternatively, an additional fee for library QC is charged per sample when researchers construct libraries within their own lab.

What lengths of index reads are supported on the NovaSeq 6000?

The standard sequence run on a NovaSeq 6000 is performed with 10 x 10 base index reads. The 10 x10 base configuration supports libraries that require dual index reads that are ten bases or shorter in length in addition to libraries with only a single index. However, NovaSeq version 1.5 sequencing kits support index read lengths with a combined total of up to 36 bases.

What is the value of unique dual indexing for libraries that are sequenced on the NovaSeq 6000?

Unique dual indexing ensures that your libraries will demultiplex with the highest potential accuracy. The NovaSeq platform enables large pools of libraries to be sequenced within individual lanes of a flow cell. Following a sequence run, the reads are sorted in a process called demultiplexing based on unique bases contained within the adapters of each library, which are sequenced during the index reads. Occasionally, an event may occur known as index hopping in which a sequence read is assigned to an incorrect sample library often based on a single, inaccurate index read. Most vendors of library preparation kits have released unique dual indexed adapters as a means to minimize the potential for index hopping. It is strongly encouraged to use unique dual indexed adapters to make sure that your libraries will demultiplex with the highest potential accuracy.

Can I provide custom primers for sequencing my libraries on the NovaSeq 6000?

Yes, the NovaSeq 6000 enables the use of custom primers for Read 1, Read 2 and the Index 1 Read using positions 5, 6, and 7 respectively in the NovaSeq cluster cartridge. Alternatively, custom primers can be combined with the standard Illumina primer using positions 24,13, and 23 respectively of the cluster cartridge. The use of custom primers is only available when the customer purchases an entire flow cell. However, sequencing with custom primers may necessitate multiple runs to define the optimal sequence for the custom primer.  

What recommendations are provided for sequencing a low diversity library?

Low diversity libraries include amplicon-based libraries (16S rRNA, CRISPR, targeted gene sequencing, etc.) and methylation analysis. These types of libraries tend to show low base diversity at each cycle of sequencing. It is best practice when sequencing on the Illumina platform that signal is detected from all colors during each sequencing cycle. To achieve this, a well base-balanced library such as PhiX is spiked into the library pool at a molar ratio of 10–15%. The addition of this balancer library provides representation of bases that would otherwise be severely limited or absent during each sequencing cycle as a means to improve base calling accuracy.

What is the run time on a NovaSeq 6000?

A sequence run on the NovaSeq 6000 requires approximately 13 to 44 hours depending on the read length.

Can the High Throughput Genomics Shared Resource provide assistance with analysis of sequence data?

The HTG Shared Resource does not provide sequence analysis services. Please contact the Bioinformatics Shared Resource for assistance with analysis at bioinformaticshelp@bio.hci.utah.edu.