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
- Please provide a price estimate for the following DNA sequencing project.
- 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 of a sequencing flow cell?
- How many lanes are on a sequencing 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 needed 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?
1. Please provide a price estimate for the following DNA sequencing project.
Example 1: Whole Genome Sequencing of 4 human DNA samples (300 million read-pairs per sample).
- Library Prep with NEBNext Ultra II FS DNA PCR-Free Library Prep Kit: (includes sample QC, library preparation and library QC/pooling)
- NovaSeq X 150x150 bp sequencing with 300 million read-pairs (90 Gb) per sample
| Description | Quantity | Unit Price | Extended Price |
|---|---|---|---|
| NEBNext Ultra II FS DNA PCR-Free Library Prep Kit | 4 | $75 | $300 |
| NovaSeq X 150x150 Sequencing (1250 M read-pairs/lane) | 1 | $1250 | $1250 |
| Total | $1,550 | ||
Example 2: RNA sequencing of 10 samples from human tissue culture cell line:
- Library Prep with NEBNext Ultra II Directional RNA with poly(A) mRNA Isolation (includes sample QC, library prep and library QC/pooling)
- NovaSeq X 150x150 bp sequencing with 20 million read-pairs per sample (200 million total read-pairs)
| Description | Quantity | Unit Price | Extended Price |
|---|---|---|---|
| NEBNext Ultra II Directional RNA Library Prep with poly(A) mRNA Isolation | 10 | $90 | $900 |
| NovaSeq X 150x150 Sequencing (100 M read-pairs) | 2 | $150 | $300 |
| Total | $1,200 | ||
2. What types of flow cells are available for the NovaSeq X?
The NovaSeq X supports three formats of flow cells; the 1.5B flow cell (1.5 billion reads split across 2 lanes), the 10B flow cell (10 billion reads split across 8 lanes) and the 25B flow cell (25 billion reads split across 8 lanes).
3. What read lengths are supported on the NovaSeq X?
Standard read lengths supported on the NovaSeq X platform include 150x150 bp, 100x100 bp and 50x50 bp.
4. What are the standard run types performed on the NovaSeq X at the HTG Shared Resource?
The standard sequence run performed at the High Throughput Genomics Shared Resource is a 150 x 150 bp sequence run on a 10B flow cell. In addition, 150 x 150 bp sequence runs on either a 1.5B or a 25B flow cells are also supported at the Shared Resource. Alternative sequence run formats are available when the customer purchases the full flow cell.
5. 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 a 1.5B flow cell (approximately 750 million read-pairs per lane), a 10B flow cell (approximately 1250 million read-pairs per lane) or a 25B flow cell (approximately 3000 million read-pairs per lane).
6. How many sequence reads are typically delivered by one lane of a sequencing flow cell?
A 1.5B flow cell should deliver approximately 1.5 billion read-pairs (750 million read-pairs per lane) according to Illumina’s specification. We typically experience 800 to 1000 million read-pairs per lane with a 2 to 5% loss of reads during demultiplex processing.
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 1400 to 1600 million read-pairs per lane with a 2 to 5% loss of reads during demultiplex processing.
A 25B flow cell should deliver approximately 25 billion read-pairs (3125 million read-pairs per lane) according to Illumina’s specification. We typically experience 3600 to 4000 million read-pairs per lane with a 2 to 5% loss of reads during demultiplex processing.
7. How many lanes are on a sequencing flow cell?
A 1.5B flow cell contains 2 lanes, whereas 10B and 25B flow cells contain 8 lanes each.
8. 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 High Throughput Genomics Shared Resource are performed with 10 x 10 base index reads.
9. 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 to 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.
10. 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. Options for sequencing these libraries include purchasing a full lane on a 1.5B, 10B or 25B flow cell or by purchasing blocks of 100 million read-pairs in a lane shared with other projects. It is important that the library is void of adapter dimer and pcr primers which could adversely affect run performance of other libraries 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.
11. 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.
12. What quantity of a sequencing library is needed 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 0.7 to 2.0 nM, depending on the library type. 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 from Roche.
13. 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 High Throughput Genomics Shared Resource when the customer purchases a full flow cell. However, the Shared Resource cannot guarantee the quality of the sequence run when custom primers are used.
14. 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 and 650 bp. Adapter sequences will add an additional 120 to 140 bp to the library size distribution.
15. 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 High Throughput Genomics 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.
16. 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 at the Huntsman Cancer Institute for assistance with analysis.
17. 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 a period of approximately 6 months. The Cancer Bioinformatics Shared Resource has enabled an option for University of Utah laboratories to mark sequencing data for long-term storage in the cloud. Please contact the Cancer Bioinformatics Shared Resource for information on creating an account if you would like to participate in this long-term storage option. Alternatively, researchers can explore other options for data storage but they should be aware that the GNomEx server will only be able to support storage for a time period of approximately 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