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Whole exome sequencing

Tomorrow’s cancer research breakthroughs are on the horizon. xGen NGS for whole exome sequencing includes offerings for routine and difficult samples, both designed to minimize efforts while maintaining key data metrics and positioning your research for limitless potential.

xGen™ NGS—made limitless.

Overview

  • Recommended products—curated for whole exome sequencing
  • A tested solution—takes you from sample to sequencer
  • Flexible sizing—available for 16 reactions or 96 reactions
  • Enzymatic fragmentation—no need for specialized equipment for library preparation
  • Individually synthesized probes—quality-controlled probes, manufactured to ISO standards, with deep, uniform coverage
  • Quality and consistency—high-quality performance metrics, displayed consistently across lots
  • Automation friendly—easy workflow that can be automated for high-throughput applications

Whole genome sequencing (WES) is a targeted next generation sequencing (NGS) approach that uses modified oligonucleotide probes to “capture” and enrich the protein coding regions (exons) in a genome. Focusing only on protein-coding exons can lower the cost and time of sequencing, as exons make up approximately 1% of the genome but contain 85% of the variants that are associated with disease [1]. WES is a practical application for various fields of research such as populations genetics and oncology research. It is also a feasible option for population genetics and discovery science, or data mining, when searching for associations, or linking genes to phenotypes [2]. The information gained from WES can provide researchers insights into germline and somatic variants within coding regions of genes.

xGen whole exome sequencing (WES) solution

For WES, samples can be prepared using multiple library preparation methods, the xGen whole exome sequencing solution employs the xGen DNA Library Prep EZ which uses enzymatic fragmentation to shear high-quality DNA samples. This kit uses ligation-based library prep, it includes enzymes for fragmentation, end-repair, and A-tailing, as well as a ligase for the addition of the xGen Stubby Adapters (included).

The xGen WES solution also calls for premixed xGen UDI Primer Pairs for indexing by PCR, which generates NGS libraries that can be directly used for hybridization capture with the xGen Exome Hyb Panel v2. This panel consists of 415,115 individually synthesized, 5’ biotinylated, oligonucleotide probes. Each probe is quality controlled and combined equimolarly to help to ensure ideal uniformity of enrichment from each panel. In an internal study, when compared to panels from other vendors, the xGen Exome Hyb Panel v2 had the highest on-target rate and mean coverage, higher depth coverage across more exons, and a more flexible workflow. Please refer to the application note, The xGen Exome Hyb Panel v2 - human exome sequencing with consistent coverage, for more information.

The xGen whole exome sequencing solution also includes xGen Hybridization Capture Core Reagents such as the xGen Hybridization and Wash Kit, xGen Universal Blockers TS, and xGen Library Amplification Primer Mix. These reagents, when paired with the xGen Hyb Panel v2, help to provide a complete, high-quality, whole exome sequencing workflow.

Icons Library_White Outline_85x85_Extraction

Extraction

Icons Library_White Outline_85x85_Library prep

LIBRARY PREP

xGen DNA Library Prep Kit EZ (16 or 96 rxn) for high quality DNA samples

xGen UDI primers (16 or 96 rxn)

Icons Library_White Outline_85x85_Sequence and analyze

Sequencing & analysis

IDT ALIGN Program

Method data

Library preparation and hybridization capture

To assess the xGen™ Exome Hyb Panel v2, twelve libraries were prepared using 100 ng of human genomic DNA (Coriell). Then, Stubby Adapters and the xGen Normalase™ Unique Dual Indexing primers (N-UDIs) were added with 6 cycles of PCR. The libraries were quantified using Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific). Indexed DNA libraries were captured and multiplexed according to the xGen Hybridization Capture of DNA Libraries Protocol. Then, to test the flexibility of the xGen Exome Hyb Panel v2 in library capture strategies, 500 ng of a DNA library was used to generate four single-plex captures and one 8-plex capture (5 total captures). Each pool underwent a 4 h hybridization capture reaction. PCR was performed on each capture using settings selected for the panel size and the number of pooled libraries in each capture (10 PCR cycles for single-plex captures; 7 PCR cycles for 8-plex captures).

Whole Exome Sequencing

The final enriched library was normalized to 4 nM and sequenced on a NextSeq® 550 instrument (Illumina®) using 2 x 150 paired end (PE) reads. The library was subsampled to 50 M total reads before analysis against the product target space in the human reference genome, hg38. Sequencing metrics were calculated using Picard analysis tools [3].

xGen DNA Library Prep EZ libraries in combination with the xGen Exome Hyb Panel v2 results in comprehensive and quality coverage

On-target rates give information regarding specificity of bases or reads aligning with a targeted region, so a higher percentage of on-target reads indicates more accurate sequencing data. Using the xGen DNA Library Prep EZ kit and the xGen Exome Hyb Panel v2 an on-target exome rate of >90% was obtained for both single-plex and 8-plex captures (Figure 1A). In addition, this workflow captured and sequenced 97% of target bases at >20X coverage depth with no noticeable drop-off in performance with 8-plex captures (Figure 1B).

Uniform coverage of both high and low GC regions indicates fewer dropouts and more complete coverage of targeted regions. To evaluate the xGen whole exome sequencing solution against this metric, Exon 1 (GC-rich) and Exon 2 (AT-rich) of the RB1 gene target space were analyzed. The sequences generated using the xGen Exome Hyb v2 panel uniformly covered both regions (Figure 2). Meaning that with the xGen whole exome sequencing solution, researchers get the uniform, comprehensive coverage, and ample read depth that is necessary for reliable variant calling.

Figure 1. xGen whole exome sequencing solution offers comprehensive coverage and read depth. Libraries were prepared using 100 ng of human genomic DNA (Coriell), and the xGen DNA Library Prep EZ Kit. Four single-plex WES captures, and one 8-plex WES capture were performed with the xGen Exome Hyb Panel v2. All captures used 500 ng of library and a 4 h hybridization reaction. The final library size was determined using a TapeStation® 4200 system and High Sensitivity D1000 ScreenTape® assay (Agilent); the final library concentration was determined using AccuClear® Ultra High Sensitivity dsDNA Quantitation kit (Biotium). Sequencing was completed on a NextSeq® 550 high-output flowcell (Illumina®) to generate 2 x 150 PE reads. Reads for all samples were down sampled to 50 M total reads. (A) Comparable flanked on-target rates are seen with both single-plex and 8-plex capture conditions. (B) 97% of target bases are covered at ≥ 20X coverage.

Figure 2. xGen™ DNA Library Prep EZ libraries in combination with the xGen Exome Hyb Panel v2 result in high quality WES. (A) Integrated Genome Viewer (IGV) [4] plots show ample coverage for both GC- and AT- rich regions of the RB1 gene.

Ordering

Resources

References

  1. Choi M, Scholl UI, et al. (2009) Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc Natl Acad Sci USA 106(45):19096–19101.
  2. Bamshad MJ, Ng SB, Bigham AW, et al. Exome sequencing as a tool for Mendelian disease gene discovery. Nat Rev Genet 2011 1211. 2011;12(11):745-755. doi:10.1038/nrg3031
  3. Picard Toolkit. Broad Institute, GitHub Repository. 2019. https://github.com/broadinstitute/picard. Broad Institute.
  4. Robinson JT, Thorvaldsdóttir H, Winckler W, et al. Integrative Genomics Viewer. Nat Biotechnol. 2011;29(1):24. doi:10.1038/NBT.1754

*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.

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