The NovaSeq 6000 is Illumina’s fastest production scale sequencing instrument. It combines two-color chemistry along with patterned flow cell technology to enable in excess of 3000 Gb of data to be sequenced on an S4 flow cell in less than two days. The NovaSeq offers the highest output and the lowest per base sequencing cost amongst Illumina instruments. It supports multiple read lengths (50, 100, 150) in paired-end format that support diverse applications including whole genome, exome, methylation, ChIP, transcriptome and 10X Genomics single cell sequencing.
Sequencing experiments on the NovaSeq platform can be ordered through the High-Throughput Genomics (HTG) Shared Resource in 100 million read-pair increments when libraries are constructed at the Resource. For additional cost-savings on large projects, the researcher can place experiment orders for full lanes or full flow cells on the NovaSeq platform. These latter two options are also supported when the researcher constructs their own libraries. Prior to sequencing, library quality control assays (Invitrogen Qubit dsDNA High Sensitivity Assay, Agilent ScreenTape Assay, Kapa qPCR) are performed to qualify individual libraries and normalize the library pool. Costs for this service are included as part of library preparation when HTG constructs the library. Alternatively, an additional fee is charged per sample or per pre-pooled sample when researchers construct libraries within their own lab.
Frequently Asked Questions
What types of flow cells are available for the NovaSeq?
Three 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).
What is the average number of read-pairs per lane on a NovaSeq 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 1.3 to 1.6 read-pairs per lane.
We can guarantee these numbers when the HTG Shared Resource constructs the library. Although we highly qualify all libraries that are sequenced on the Illumina platform, we are unable to guarantee these numbers when individual researchers construct their own sequencing libraries due to variability in library quality that is outside of our control.
What run types are supported on the NovaSeq platform at the HTG Shared Resource?
The HTG Shared Resource supports 2 x 150 bp sequence runs on S4 flow cells and 2 x 50 bp runs on S2 flow cells. Please contact the Shared Resource for inquiries regarding alternative flow cell types or read lengths.
What options of sequencing output are available when placing a NovaSeq experiment order?
Sequencing experiments on the NovaSeq platform can be ordered through the High-Throughput Genomics Shared Resource in 100 million read-pair increments when libraries are constructed at the Resource. For additional cost-savings on large projects, the researcher can place experiment orders for full lanes or full flow cells on the NovaSeq platform. These latter two options are also supported when the researcher constructs their own libraries.
What is the run time on a NovaSeq?
A 2 x 150 bp run on a NovaSeq S4 flow cell requires approximately 44 hours of run time. A 2 x 50 bp run on a NovaSeq S2 flow cell requires approximately 16 hours of run time.
Can the HTG Shared Resource provide assistance with analysis of sequence data?
The High-Throughput Genomic Shared Resource does not provide sequence analysis services. Please contact the Bioinformatics Shared Resource (email@example.com) for assistance.
Can I construct my own libraries for sequence analysis on the NovaSeq?
Libraries constructed by individual researchers can be sequenced on the NovaSeq. Experiment orders can be placed for either full lanes on the NovaSeq or full flow cells. Unfortunately, we are unable to support smaller volumes of sequencing output at this time for libraries constructed by individual labs
What quantity of a sequencing library is required for sequence analysis on a NovaSeq when a researcher constructs their own libraries?
Each lane on a NovaSeq S4 flow cell requires a 30 ul volume of a 1.15 nM pool of libraries. An S2 flow cell requires a 20 L volume.
Should I be concerned if adapter dimer products are present in my sequencing library?
The NovaSeq platform is very sensitive to adapter dimer representation in sequencing libraries. A library that contains 5% adapter dimer may be expected to yield as much as 60% adapter-only sequence reads. It is important for researchers that construct their own sequencing libraries to select against the presence of adapter dimers during the library purification process.
How do I know if my library contains adapter dimers?
Adapter dimers exhibit a size distribution of approximately 140 to 160 bp in libraries that include dual indexed adapters. Adapter dimers in single indexed libraries are approximately 120 to 130 bp.
Can I provide custom primers for sequencing my libraries on the NovaSeq?
The use of custom primers is not supported on the NovaSeq.
What is the optimal insert size for libraries that are sequenced on the NovaSeq?
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 100 and 500 bp. Adapter sequences will add an additional 140 bp to the library.
How is the library’s quality assessed prior to sequence analysis on the NovaSeq?
Quality control assays are performed to validate libraries prior to sequence analysis on the NovaSeq. These assays include the following: Qubit dsDNA High Sensitivity Assay (library concentration), Agilent ScreenTape Assay (size distribution), MiSeq Nano sequence run (normalize library representation within a pool), and qPCR of pooled libraries to calculate and adjust and molarity of library pools in preparation for applying to a NovaSeq flow cell. Costs for these quality control assays are included as part of library preparation when the HTG Shared Resource constructs the library. Alternatively, an additional fee is charged per sample when researchers construct libraries within their lab.
What length of index reads are supported on the NovaSeq?
The NovaSeq supports 2 x 10 base index reads. The 2 x 10 base configuration supports libraries which require dual index reads that are ten bases or shorter in length in addition to libraries with only a single index. However, we highly discourage sequencing libraries with a single index on the NovaSeq system due to the potential of unresolved barcode hopping that can be experienced with such libraries.
What is the value of unique dual indexes for libraries sequenced on the NovaSeq?
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 sequences contained within the adapters of each library which are identified during the index reads. Index hopping is an event which results in the assignment of sequence reads to an incorrect sample library. Many 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.
What recommendations are available for sequencing a low diversity library?
Examples of low diversity libraries include bisulfite-treated DNA libraries, 16S rRNA libraries, CRISPR libraries, and single amplicon libraries. In each of these cases, one or more nucleotides is significantly under-represented during each cycle of the sequence run which can negatively impact accurate base calling within a sequence lane. To overcome the impact of low diversity, a balanced library such as the Illumina PhiX v3 library, can be added to the lane at a molarity such that it will represent approximately 15-20% of the sequence reads within the lane.