Targeted deep resequencing of the human cancer genome using next-generation technologies

Abstract

Next-generation sequencing technologies have revolutionized our ability to identify genetic variants, either germline or somatic point mutations, that occur in cancer. Parallelization and miniaturization of DNA sequencing enables massive data throughput and for the first time, large-scale, nucleotide resolution views of cancer genomes can be achieved. Systematic, large-scale sequencing surveys have revealed that the genetic spectrum of mutations in cancers appears to be highly complex with numerous low frequency bystander somatic variations, and a limited number of common, frequently mutated genes. Large sample sizes and deeper resequencing are much needed in resolving clinical and biological relevance of the mutations as well as in detecting somatic variants in heterogeneous samples and cancer cell sub-populations. However, even with the next-generation sequencing technologies, the overwhelming size of the human genome and need for very high fold coverage represents a major challenge for up-scaling cancer genome sequencing projects. Assays to target, capture, enrich or partition disease-specific regions of the genome offer immediate solutions for reducing the complexity of the sequencing libraries. Integration of targeted DNA capture assays and next-generation deep resequencing improves the ability to identify clinically and biologically relevant mutations.

Figure 1

Solid-phase target DNA capture. Oligonucleotides, bacterial artificial clones or PCR products associated with the targeted genomic regions are immobilized on solid surface, either on microarray glass slide or nitrocellulose filter. Target capture is executed by DNA hybridization.

Figure 2

Multiplex PCR and solution-phase target DNA capture. A) Nested Patch PCR utilizes two sets of primers and subsequent PCR reactions to amplify specific target regions. B) The Gene-Collector assay is based on collector oligonucelotide probe, which selectively circularizes correctly amplified PCR products. C) Targeted genomic circularization technology uses “Vector” and “Targeting” oligonucleotides to circularize genomic DNA fragments that have been digested with restriction enzymes. D) Padlock probe and E) Molecular inversion probe assays induce circular DNA molecules by cap filling between specific recognition sites.

Figure 3

Hybrid-phase target DNA capture. Microarray production platform is used to generate oligo-nucleotides that are further processed to biotinylated RNA baits. Baits are hybridized with genomic DNA fragments that have been ligated with sequencing adaptors. Streptavidin coated beads are then used to collect selected DNA fragments.

Figure 4

Experimental designs of 1 Tb sequencing projects. Experimental dimensions of next-generation sequencing projects of the whole cancer genome (green), protein coding exons (blue), cancer genes (red), 25 target genes (purple) and mutation biomarkers from circulating tumor cells (yellow) are illustrated in the figure. When sample number increase less stringent criteria for sample inclusion can be maintained, suggesting that more sequencing depth is required for large sample size studies. Moreover, sample multiplexing necessitates sequencing barcode sequences, which also is reflected in the required sequencing depth in up-scaled projects.