A Flexible Approach for Highly Multiplexed Candidate Gene Targeted Resequencing

Abstract

We have developed an integrated strategy for targeted resequencing and analysis of gene subsets from the human exome for variants. Our capture technology is geared towards resequencing gene subsets substantially larger than can be done efficiently with simplex or multiplex PCR but smaller in scale than exome sequencing. We describe all the steps from the initial capture assay to single nucleotide variant (SNV) discovery. The capture methodology uses in-solution 80-mer oligonucleotides. To provide optimal flexibility in choosing human gene targets, we designed an in silico set of oligonucleotides, the Human OligoExome, that covers the gene exons annotated by the Consensus Coding Sequencing Project (CCDS). This resource is openly available as an Internet accessible database where one can download capture oligonucleotides sequences for any CCDS gene and design custom capture assays. Using this resource, we demonstrated the flexibility of this assay by custom designing capture assays ranging from 10 to over 100 gene targets with total capture sizes from over 100 Kilobases to nearly one Megabase. We established a method to reduce capture variability and incorporated indexing schemes to increase sample throughput. Our approach has multiple applications that include but are not limited to population targeted resequencing studies of specific gene subsets, validation of variants discovered in whole genome sequencing surveys and possible diagnostic analysis of disease gene subsets. We also present a cost analysis demonstrating its cost-effectiveness for large population studies.

The Human OligoGenome Resource: a database of oligonucleotide capture probes for resequencing target regions across the human genome

“…These rules correspond to the empirical best practices empirically determined by Natsoulis et al (3): Capture arms are 20 bp in length;The sequences in a pair of capture arms must have the same polarity with respect to the reference genome;3′ capture arms must be flush to a restriction site; andThe maximum size of a DNA molecule targeted by a capture oligonucleotide is 800 bp and the minimum size is 100 bp.…”

“…This vector provides the universal primer sequences necessary for downstream amplification. Previously, we designed a set of capture oligonucleotides spanning the human exome (http://oligoexome.stanford.edu) and demonstrated that customized capture assays could be easily developed using this resource (3).

Figure 1.Schema for target-specific capture and amplification by selective genomic circularization.

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Figure 1.Schema for target-specific capture and amplification by selective genomic circularization. This schema for the Natsoulis et al (3) capture protocol describes the major steps for conducting capture and amplification of a target region. The light blue squiggles at the top of the figure indicate restriction enzyme recognition sites that are cut by the addition of a single restriction enzyme.

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“…Using our previous experience in designing and implementing assays, we improved the design method to avoid issues which decrease capture efficiency (3). This unique resource has extensive utility given that it provides coverage for capture targets beyond the 3% of the coding region portion (e.g.…”

Efficient targeted resequencing of human germline and cancer genomes by oligonucleotide-selective sequencing

“…Targeted resequencing also has the potential to become an important diagnostic tool. Academic and commercial groups have developed a variety of capture methods for enriching or selectively amplifying subsets of the genome for targeted resequencing 4–10 . These approaches are generally based on either PCR amplification, target-specific DNA circularization or hybridization-based selection of DNA targets.…”