- Integrated library preparation and exome enrichment for highly uniform coverage of coding regions
- Flexible, scalable, benchtop gene sequencing system for superior data quality
- The on-board data analysis pipeline has an award-winning performance in detecting common mutations and rare somatic variants
Brief introduction
The NextSeq 1000/1000-CN and NextSeq 2000/2000-CN Series Sequencing System exome sequencing solutions (collectively, the "NextSeq 1000/2000 Exon Sequencing Solutions") provide an integrated workflow from DNA library preparation to analysis of sequencing results, enabling researchers to explore protein-coding (exon) regions of the genome. The solution is powered by Illumina's industry-leading next-generation sequencing (NGS) technology and optimized sequencing-by-synthesis (SBS) chemistry, XLEAP-SBS™ chemistry, to deliver superior sequencing data. This protocol provides highly accurate exome coverage to identify true coding variants for a wide range of applications, including population genetics, genetic disease research, and cancer research. In the NextSeq 1000/2000 exome sequencing protocol, workflows can be integrated from library preparation to exon enrichment to one-click sequencing and fast and accurate data analysis on a single benchtop sequencing (Figure 1). Flexible and efficient exome sequencing can be done with minimal hands-on time.
Simple, efficient workflow
Figure 1: NextSeq 1000/2000 Exome Sequencing Workflow—a simple, integrated NGS sequencing solution that delivers highly accurate exome sequencing data. Working hours vary depending on the type of experiment and assay.
The NextSeq 1000/2000 Exome Sequencing Solution provides a streamlined, integrated workflow that enables researchers to be more productive. Library preparation and exome enrichment were performed using library kits (e.g., Illumina DNA Prep with Exome 2.5 Enrichment), the prepared libraries were loaded into flow cells, and then loaded into the NextSeq 1000/1000-CN and NextSeq 2000/2000-CN Series Sequencing Systems (collectively referred to as the "NextSeq 1000/2000 Series Sequencing Systems") for sequencing (Figure 2). The NextSeq 1000/2000 Series Sequencing Systems are available in a variety of sequencing flow cell configurations, allowing researchers to scale their exome studies to their needs.
Figure 2: NextSeq 1000/2000 Series Sequencing Systems – NextSeq 1000/2000 Series Sequencing Systems are equipped with XLEAP-SBS chemistry technology and an on-board secondary data analysis module to integrate sequencing workflows.
All-in-one
Library preparation and exome enrichment
The Illumina DNAPrep with Exome 2.5 Enrichment Kit for DNA library preparation using the fast, bead-bound transposase chemistry and exome enrichment with the Illumina Exome 2.5 Panel enable researchers to quickly identify true coding variants. Only 10 ng of input is required to provide full exome coverage, which allows laboratories to analyze valuable DNA samples, while also providing highly uniform coverage and enrichment. Highly sensitive detection of low-frequency variants helps laboratories accurately identify true coding variants and rare somatic mutations.
On-Bead Tagmentation eliminates the mechanical cutting step and streamlines the workflow to approximately 6 hours of total time compared to less than 2 hours of hands-on time. With Illumina DNA Prep with Enrichment, researchers can select panel content from multiple vendors, including Illumina, Twist, and IDT. This means researchers can retain the workflow and data quality benefits of Illumina DNA Prep with Enrichment across multiple exome panels.
NextSeq 1000/2000
Serial sequencing system
The powerful and flexible performance of the NextSeq 1000/2000 Series Sequencing Systems streamlines exome sequencing workflows. The total time to load and start up the system was less than 10 minutes. Using NextSeq 2000 P4 Reagents, up to 41 samples can be sequenced in less than 34 hours at 2×100 bp. *
The NextSeq 1000/2000 Series Sequencing Systems are compatible with a range of library preparation kits from Illumina and third-party, providing flexibility across applications. Researchers were able to easily switch between multiple sequencing projects, such as exome, population cell, and single-cell RNA sequencing (RNA-Seq), and other applications (Table 2).
*Sequencing throughput may vary depending on a number of factors, including the size of the exome panel used and the library preparation kit.
Achieve "true coding variant" calling
Calling a true coding variant refers to the accurate detection of a base that is different from the congruent sequence within the coding region. It is not a false positive (the variant is detected, but it is not really present) or a false negative (the variant is really present, but it is not detected). Systems with a high false-positive rate require extensive downstream validation, increasing cost and experimental time. Systems with high false-negative results fail to detect potentially important results because they tend to be located in areas with high repetition or containing homomer extensions. Achieving true coding variant calling requires high-quality library preparation and enrichment, superior sequencing accuracy, and secondary analysis accuracy, which is exactly what Illumina's NextSeq 1000/2000 Series Sequencing Systems with XLEAP-SBS chemistry provide.
Explore with XLEAP-SBS chemistry
Powered by XLEAP-SBS chemistry, Illumina's fastest, highest-quality, and most reliable sequencing chemistry to date, the Nextseq 1000/2000 Series sequencing systems are the highest-throughput, lowest-cost single-sample sequencing systems of any Illumina benchtop sequencer. At 2×100 bp, the sequencer achieves excellent accuracy, achieving ≥ 85% base scores above Q30† (Table 2), with high accuracy even in challenging regions such as GC-rich regions or homopolymers, allowing for more true coding variants. The extremely low false positive and false negative rates greatly reduce the time and cost of downstream validation. The NextSeq 1000/2000 Series Sequencing Systems provide superior data quality and are ideal for comprehensive exome studies.
The NextSeq 1000/2000 Series Sequencing Systems feature proven next-generation sequencing technology that supports all Illumina sequencing systems, enabling researchers to compare and integrate data generated across systems. For example, exome sequencing data from the NextSeq 1000/2000 Series Sequencing Systems can be integrated with data from targeted panel assays based on tracking studies or large-scale exome sequencing data running on the NovaSeq™ X Sequencing System (Table 3).
USE DRAGEN SECONDARY ANALYSIS
Simplify your analysis
The NextSeq 1000/2000 Series Sequencing Systems are equipped with the DRAGEN Secondary Analysis Module, which enables laboratories to perform accurate, comprehensive, and efficient data analysis in exome sequencing. ‡This data analytics solution§ uses hardware-accelerated, optimized algorithms that have been approved by the FDA Truth Challenge to help users reduce reliance on external informatics experts and overcome data analysis bottlenecks.
The NextSeq 1000/2000 Series Sequencing Systems perform data analysis using the DRAGEN Enrichment process, resulting in accurate variant calls within two hours of a sequencing run. This method has excellent accuracy for both germ and somatic genome alignment and small variant calling6-8.
‡ NextSeq 1000/2000 Series Sequencing Systems include DRAGEN hardware. The instrument comes with a DRAGEN license and does not need to be purchased separately.
§ In the 2020 PrecisionFDA Truth Challenge v2, the DRAGEN Secondary Analysis received the "Best of Perform" award for sequencing analysis of hard-to-map regions of sequencing data and all reference regions7,8.
Table 2: Performance parameters of the NextSeq 1000/2000 Series Sequencing Systems for exome sequencing
a. Throughput data were obtained experimentally on a single flow cell using an Illumina PhiX control library at an appropriate cluster density.
b. Run time calculations include cluster generation, sequencing, and base calling on the NextSeq 1000/2000 Series Sequencing System.
c. Mass score calculations are based on Illumina PhiX QC libraries. Performance may vary depending on library type and quality, insert size, loading concentration, and other experimental factors. Base ratios above Q30 are based on the average of the entire run.
XLEAP-SBS reagents for P1, P2, and P3 flow cells will be available in Q2 2024.
e. P3 and P4 reagents are only available on the NextSeq 2000/2000-CN Gene Sequencer.
Table 3: Exome sequencing throughput of Illumina's different gene sequencers
a. Exome count calculations assume approximately 8Gb per sample, resulting in 100× coverage. Throughput can vary depending on a number of factors, including the size of the exome panel used and the library prep kit.
b. P3 and P4 reagents are only available on 2000/2000-CN gene sequencers.
c. The NovaSeq X Plus Gene Sequencer supports single or dual flow cell operation. The NovaSeq X Gene Sequencer supports single-flow cell operation only.
d. Up to 384 unique dual-label sequences are available. For the NovaSeq X Series, independent channel loading enables multiplexing of more samples.
The NextSeq 1000/2000 Exome Sequencing Solution combines the power, speed, and flexibility of the NextSeq 1000/2000 Series Sequencing Systems with high-quality library preparation and enrichment options and user-friendly analysis software to provide an integrated, flexible sequencing solution for coding variant call.
- NIH National Library of Medicine. RefSeq: NcBi Reference Sequence database. ncbi.nlm.nih.gov/refseq. Updated July 18,2023. accessed august 25, 2023.
- The GeNcOde Project. GeNcOde: encyclopedia of genes and gene variants. gencodegenes.org/. accessed august 25, 2023.
- NcBi website. consensus coding sequences (ccdS) database.ncbi.nlm.nih.gov/projects/CCDS/CcdsBrowse.cgi.Updated November 9, 2022. accessed august 25, 2023.
- University of california, Santa cruz Genome Browser. UcSc Known Genes. genome.ucsc.edu/. Updated august 18, 2023. accessed august 25, 2023.
- NiH National Library of Medicine. clinVar database. ncbi.nlm.nih.gov/clinvar. Updated August 28, 2023. Accessed August 28,2023.
- illumina. Accuracy improvements in germline small variant callingwith the DRAGEN Platform. accessed august 25, 2023.
- PrecisionFda website. truth challenge V2: calling variants from short and long reads in difficult-to-map regions. precision.fda.gov/challenges/10. Accessed August 25, 2023.
- Mehio R, Ruehle M, catreux S, et al. dRaGeN Wins at Precision-Fda truth challenge V2 Showcase accuracy Gains from altaware Mapping and Graph Reference Genomes. illumina.com/ science/genomics-research/dragen-wins-precisionfda- challengeshowcase-accuracy-gains.html. Accessed August 25, 2023.
For research use only and not for diagnostic use.