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

Abstract

We describe an approach for targeted genome resequencing, called oligonucleotide-selective sequencing (OS-Seq), in which we modify the immobilized lawn of oligonucleotide primers of a next-generation DNA sequencer to function as both a capture and sequencing substrate. We apply OS-Seq to resequence the exons of either 10 or 344 cancer genes from human DNA samples. In our assessment of capture performance, >87% of the captured sequence originated from the intended target region with sequencing coverage falling within a tenfold range for a majority of all targets. Single nucleotide variants (SNVs) called from OS-Seq data agreed with >95% of variants obtained from whole-genome sequencing of the same individual. We also demonstrate mutation discovery from a colorectal cancer tumor sample matched with normal tissue. Overall, we show the robust performance and utility of OS-Seq for the resequencing analysis of human germline and cancer genomes.

Figure 1

Overview of OS-Seq. Capture of targets, processing and sequencing are performed on an Illumina next-generation sequencer. Reads originating from each primer probe are target and strand specific. Shown here is the median coverage profile for OS-Seq-366. For step 1, target-specific oligonucleotides are used to modify flow cell primers to create ‘primer probes’. Hybridized oligonucleotides are used as a template for DNA polymerase and D primers are extended. After denaturing, target-specific primer probes are randomly immobilized on the flow cell. For step 2, genomic targets in a single-adaptor library are captured using primer probes. These adapters incorporate sites for sequencing primers and immobilized flow cell primers. Targets in the single-adaptor library are captured during a high-heat hybridization step to their complementary primer probes. Captured single-adaptor library fragments are used as a template for DNA polymerase, and primer probes are extended. Denaturation releases template DNA from immobilized targets. For step 3, immobilized captured targets are rendered to be compatible for DNA sequencing. During a low-heat hybridization step the single-adaptor tails of the immobilized targets hybridize to type C primers on the flow cell surface, which stabilizes a bridge structure. The 3′ ends of immobilized targets and C primers are extended using DNA polymerase, which creates two molecules capable of bridge PCR. After denaturation, bridge amplification and cluster processing, paired-end sequencing can be conducted.

Figure 2

Targeted sequencing coverage profile along the KRAS gene from the OS-Seq-366 assay. Base positions relative to the start of exon 1 are presented on the x axis and KRAS exons are marked in red on the x axis. Sequencing fold coverage is listed on the y axis.

Figure 3

Coverage assessment of OS-Seq. Uniformity assessment of primer-probe yields within column- and array-synthesized oligonucleotides. We compared the uniformity of capture between column-synthesized (blue, n = 366) and array-synthesized (red, n = 11,742) oligonucleotides. On the x axis, oligonucleotides are sorted by sequence capture yields, on the y axis is the normalized primer probe yield. To calculate normalized yield, we divided the yield of each oligonucleotide by the median yield from all oligonucleotides.