Next Generation Sequencing
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ve-SEQ: An improved approach to high throughput, whole genome viral sequencing

xGen® Lockdown® Probes provide deep coverage of multiple HCV genotypes in clinical samples

Bonsall D, Ansari MA, et al. (2015) ve-SEQ: Robust, unbiased enrichment for streamlined detection and whole-genome sequencing of HCV and other highly diverse pathogens. F1000 Research 4:1062.

Background

Hepatitis C virus (HCV) is prevalent in 2.8% of the global population and is currently incurable, largely due to its extensive genetic diversity. While antiviral drug combination therapies have improved treatment outcomes, they are ineffective against certain HCV genotypes and are also extremely expensive. Recent clinical trials assessing resistant-associated variants (RAVs) suggest treatment efficacy largely depends on the genetic background of the HCV virus. Therefore, efficient methods for whole genome viral sequencing are in high demand. Such methods could offer insights into the relevance of low frequency variants, the impact of mutation combinations on viral fitness, and the drug susceptibilities of specific viral genotypes.

In this paper, the authors introduce a novel approach for high throughput, whole genome HCV sequencing. They demonstrate how their method, ve-SEQ, improves on conventional viral sequencing methods such as PCR amplification and RNA-seq based metagenomics.

Experiment

Bonsall et al. began by evaluating an RNA-seq, metagenomic approach to HCV sequencing. They constructed indexed sequencing libraries from plasma RNA of HCV-infected patients and mapped the relationship between viral load (VL) and the number of HCV reads through sequencing on the Illumina HiSeq 2500.

The researchers then performed their ve-SEQ method, comparing its sequencing efficiency to the conventional RNA-seq method. In their ve-SEQ approach, they enriched the previously-sequenced pool of libraries with an optimized capture panel made from IDT xGen® Lockdown® Probes. The panel contained 4 sets of 155–157 biotinylated probes, each 120 nt, and provided capture of 4 HCV genotypes.

Initially, they used this capture panel to investigate how varying probe-target sequence identities can affect ve-SEQ success. The information gathered from these investigations prompted the researchers to develop an algorithm for probe design that allowed them to build a comprehensive HCV panel for use in a clinical setting.

Results

The researchers found that the RNA-seq based, metagenomic approach generated 1% total HCV sequence yield from samples with high viral load (VL). They note how this approach, while unbiased, is costly and insensitive to samples with low to mid-range VL. With their ve-SEQ method, however, the group was able to generate linear detection of VLs down to 1000 IU/mL—more than 10X lower than by regular RNA-seq. In addition, ve-SEQ (with xGen probes) led to a 224X increase in total HCV reads, including 1000X enrichment for samples with low to mid-range VL, and nearly 100% HCV sequence content for high VL samples.

Through investigation of probe-target sequence identity effects on ve-SEQ success, they found that near-maximal enrichment is achievable if the sequence of a sample segment and its closest matching probe is ≥80% identical. This finding prompted the researchers to implement an algorithm for optimizing probe design, which resulted in the assembly of a comprehensive HCV panel representing 6 of the 7 currently recognized HCV genotypes.

The ve-SEQ based, optimized panel ultimately offered robust, unbiased detection of resistance-associated variants (RAVs) in a clinical setting. The researchers successfully monitored viral re-emergence in patients undergoing antiviral drug treatments, and were able to identify known drug resistant alleles within these new populations. Overall, the group’s findings support ve-SEQ as a cost-effective improvement over other high throughput approaches to whole genome viral sequencing, and suggest similar methods could be applied to other pathogens.

Get technical assistance for your NGS research

The authors of this paper obtained technical advice from IDT’s NGS support group. The expertise they received prompted them to acknowledge Nick Downey, one of our Scientific Application Specialists, as a contributor to their research.

If you have questions regarding the design of your NGS research, contact our support team at xGen@idtdna.com.

Product focus: Target capture reagents from IDT


xGen® Lockdown® Probes

xGen Lockdown Probes are individually synthesized, quality controlled, and normalized hybridization probes that offer:

  • Sensitive detection of SNPs, indels, CNV, LOH, and translocations
  • GMP compliance for clinical and diagnostics research
  • Flexibility to augment existing panels or create completely custom panels
  • Quick delivery

Discover more about xGen Lockdown Probes


xGen® Lockdown® Panels

xGen Lockdown Panels are preconfigured, validated, and stocked pools of xGen Lockdown Probes for targeted next generation sequencing of defined gene families:

  • xGen Exome Research Panel
  • xGen Acute Myeloid Leukemia Panel
  • xGen Pan-Cancer Panel
  • xGen Inherited Diseases Panel

Discover more about xGen Lockdown Panels


xGen® Lockdown Reagents Hybridization and Wash Kit

Achieve uniform coverage with hybridization and wash buffers that are optimized for target enrichment using xGen Lockdown Probes and Panels. A short, 4-hour hybridization protocol generates results quickly.

Discover more about xGen Lockdown Reagents.


xGen® Blocking Oligos

xGen Universal Blocking Oligos for single- or dual-index adapters used with common sequencing platforms improve on-target performance for multiplexed samples by reducing adapter participation in hybridization enrichment. Custom adapters can be manufactured for other barcodes or to meet the needs of customers who require specific modifications or services to improve performance in unique applications.

Discover more about xGen Blocking Oligos

Additional reading

  • Citation: Gene panels vs. gene by gene analysis for assessing disease risk—Read about a comparison of NGS-based gene panels and traditional testing data for diagnostic use and disease risk assessment in hereditary breast and ovarian cancer. xGen Lockdown Probes (IDT) were used to rescue drop-out regions of SureSelect (Agilent) probe panels.
  • Citation: Next generation sequencing for risk assessment in acute myeloid leukemia—See how scientists apply next generation sequencing (NGS) techniques with patients diagnosed with AML to identify associations between specific mutations and disease outcome. The xGen® AML Cancer Panel v1.0 was used to enrich for 264 commonly mutated genes in AML.
  • Article: How important are those NGS metrics?—Of the many metrics used in evaluating target capture data for NGS applications, read about which ones our researchers consider important for evaluating performance of target enrichment panels.  

Nolan Speicher is a scientific writing intern at IDT.

© 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.


xGen® Blocking Oligos

Adapter blocking oligos increase the number of on-target reads by preventing non-specific binding during hybridization.

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