The NovaSeq X offers the highest output and the lowest per base sequencing cost amongst Illumina’s sequencing instruments. It supports multiple read lengths (50, 100, 150) in paired-end format that support diverse sequencing applications including whole genome, exome, methylation, ChIP-seq, transcriptome and single cell analysis. Sequencing experiments on the NovaSeq platform can be ordered at the High-Throughput Genomics (HTG) Shared Resource in blocks of 100 million read-pairs. Additional cost savings on larger projects are available by ordering full lanes on a 10B flow cell (1250 million read-pairs) or a 25B flow cell (3000 million read-pairs).
Prior to sequencing, library quality control assays (Qubit dsDNA Assay, Agilent DNA ScreenTape Assay, Kapa Library Quantification by qPCR) are performed to qualify libraries and normalize the library pool. The cost for this service is included as part of library preparation when the libraries are constructed at the HTG Shared Resource. Alternatively, an additional fee is charged per sample (or per pre-pooled samples) when researchers construct libraries within their own lab.
Frequently Asked Questions
- What types of flow cells are available for the NovaSeq X?
- What read lengths are supported on the NovaSeq X?
- What are the standard run types performed on the NovaSeq X at the HTG Shared Resource?
- What options of sequencing output are available when placing an experiment order for the NovaSeq X platform?
- How many sequence reads are typically delivered by one lane on a 10B flow cell?
- What length of index reads are supported on the NovaSeq?
- What recommendations are available for sequencing a low diversity library?
- Can I construct my own libraries for sequence analysis on the NovaSeq X?
- Should I be concerned if adapter dimer products are present in my sequencing library?
- What quantity of a sequencing library is required for sequence analysis on a NovaSeq X when a researcher constructs their own libraries?
- Can I provide custom primers for sequencing my libraries on the NovaSeq X?
- What is the optimal insert size for libraries that are sequenced on the NovaSeq X?
- How is library quality assessed prior to sequence analysis on the NovaSeq X?
- Can the HTG Shared Resource provide assistance with analysis of sequence data?
- How long will sequencing data from my experiment be available for download on the GNomEx server?
3. What are the standard run types performed on the NovaSeq X at the HTG Shared Resource?
The standard sequence run performed at the HTG Shared Resource is a 150 x 150 bp sequence run on a 10B flow cell. In addition, 150 x 150 bp sequence runs on a 25B flow cells are also supported. Alternative sequence run formats are available when the customer purchases the full flow cell.
4. What options of sequencing output are available when placing an experiment order for the NovaSeq X platform?
Sequence reads on the NovaSeq X can be ordered in blocks of 100 million read-pairs (30 Gb) on a 150x150 bp run. Alternatively, additional cost savings on large projects is available by ordering full lanes on an 10B flow cell (approximately 1250 million read-pairs per lane) or full lanes on the 25B flow cell (approximately 3000 million read-pairs per lane).
5. How many sequence reads are typically delivered by one lane on a 10B flow cell?
A 10B flow cell should deliver approximately 10 billion read-pairs (1250 million read-pairs per lane) according to Illumina’s specification. We typically experience 1200 to 1500 million read-pairs per lane with a 3–6% loss of reads during demultiplex processing of the sequence reads from a lane.
6. What length of index reads are supported on the NovaSeq?
A sequence kit for the NovaSeq X platform contains sufficient sequencing reagents to enable the sequencing of the library insert and up to 36 additional bases representing index reads and unique molecular identifiers (UMIs). Standard runs at the HTG Shared Resource are performed with 20 x 10 base index reads.
7. What recommendations are available for sequencing a low diversity library?
Examples of low diversity libraries include Methyl-seq libraries in which non-methylated C residues have been converted to T or amplicon libraries including 16S rRNA, CRISPR and targeted gene libraries. In each of these cases, one or more nucleotides are under-represented during each cycle of the sequencing process. The NovaSeq X uses 2-channel sequencing chemistry which performs best when all four DNA bases are represented during each sequencing cycle. A failure to adequately represent each base can negatively impact quality scores and accurate base calling within the sequence lane.
To overcome the impact of low diversity libraries, a base-balanced library such as the Illumina PhiX v3 library can be added to the lane at a molarity such that it will represent approximately 10–15% of the sequence reads within the lane. This balancer library will then enable sufficient representation of all four bases during each cycle of the sequence run.
8. Can I construct my own libraries for sequence analysis on the NovaSeq X?
Libraries constructed by individual researchers can be sequenced on the NovaSeq X. These libraries will be qualified by library quality control assays including Qubit dsDNA High Sensitivity assay, Agilent DNA ScreenTape assay and qPCR using the Kapa Library Quantification kit. If we have concerns on library quality, we will contact you.
It is important that the library is void of adapter dimer and pcr primers which could adversely affect run performance of other samples that are sequenced in the same lane. To avoid these products in your library, we recommend performing two successive purifications of your library following pcr amplification. Alternatively, we are willing to re-purify your libraries for an additional fee if necessary.
Options for sequencing these libraries include purchasing a full lane (1250 million reads on a 10B flow cell) or purchasing blocks of 100 million read-pairs in a lane shared with other projects.
9. Should I be concerned if adapter dimer products are present in my sequencing library?
Adapter dimer products, which typically appear as 120 to 140 bp bands on an Agilent DNA ScreenTape Assay, are able to hybridize to Illumina sequencing flow cells more efficiently than library molecules that contain a DNA insert. A library that is represented by 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 eliminate adapter-dimer products from their final library preparation.
10. What quantity of a sequencing library is required for sequence analysis on a NovaSeq X when a researcher constructs their own libraries?
Sequence runs performed on a NovaSeq X 10B flow cell requires a 34 ul volume of each library pool that is adjusted to a molarity of 1.0–2.5 nM. Note that different library prep kits will require optimal loading on a flow cell to be performed at different molarities. The molarity of sequencing libraries is established by qPCR using the KAPA Library Quantification Kit.
11. Can I provide custom primers for sequencing my libraries on the NovaSeq X?
The use of custom primers on the NovaSeq X requires the custom primer to be distributed across all lanes of the flow cell. Sequence runs with custom primers can be supported at the HTG Shared Resource when the customer purchases a full flow cell. However, theHTG Shared Resource cannot guarantee the quality of the sequence run when custom primers are used.
12. What is the optimal insert size for libraries that are sequenced on the NovaSeq X?
The NovaSeq X flow cell contains 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 50–650 bp. Adapter sequences will add an additional 120 to 140 bp to the library size distribution.
13. How is library quality assessed prior to sequence analysis on the NovaSeq X?
Quality control assays are performed to validate libraries prior to sequence analysis on the NovaSeq X. These assays include the following: Qubit dsDNA High Sensitivity Assay (library concentration), Agilent DNA ScreenTape Assay (size distribution), and qPCR with the Kapa Library Quantification Kit to calculate and normalize the molarity of individual libraries when preparing library pools to apply to a NovaSeq X flow cell.
The cost for these quality control assays is included as part of library preparation when the HTG Shared Resource constructs the library. Alternatively, an additional fee is charged per sample (or pre-pooled sample) when researchers construct libraries within their own lab.
14. Can the HTG Shared Resource provide assistance with analysis of sequence data?
The HTG Shared Resource does not provide sequence analysis services. Please contact the Cancer Bioinformatics Shared Resource for assistance with analysis at bioinformaticshelp@bio.hci.utah.edu.
15. How long will sequencing data from my experiment be available for download on the GNomEx server?
Sequencing data will be available on the GNomEx server for approximately six months. The Cancer Bioinformatics Shared Resource has enabled an option for University of Utah laboratories to migrate sequencing data for long-term cloud storage. Please contact the Cancer Bioinformatics Shared Resource for additional information on long-term storage options. Alternatively, researchers can explore other options for data storage but they should be aware that the GNomEx server is unable to support a solution in excess of six months.
Contact Us
High-Throughput Genomics Director
Brian K. Dalley, PhD
High-Throughput Genomics Associate Director
Opal Allen, PhD
Governance
HCI Senior Director Oversight
Alana Welm, PhD
Faculty Advisory Committee Chair
Katherine Varley, PhD
Faculty Advisory Committee Members
Richard Clark, PhD
Jason Gertz, PhD
Christopher Gregg, PhD
Mei Koh, PhD
Philip Moos, PhD
Andrew Post, MD, PhD
Sean Tavtigian, PhD
Joseph Yost, PhD