Exome sequencing: the sweet spot before whole genomes

Abstract

The development of massively parallel sequencing technologies, coupled with new massively parallel DNA enrichment technologies (genomic capture), has allowed the sequencing of targeted regions of the human genome in rapidly increasing numbers of samples. Genomic capture can target specific areas in the genome, including genes of interest and linkage regions, but this limits the study to what is already known. Exome capture allows an unbiased investigation of the complete protein-coding regions in the genome. Researchers can use exome capture to focus on a critical part of the human genome, allowing larger numbers of samples than are currently practical with whole-genome sequencing. In this review, we briefly describe some of the methodologies currently used for genomic and exome capture and highlight recent applications of this technology.

Figure 1.

Illustration of different capture methods. Light blue bars represent desired genomic sequence, red bars represent unwanted sequence. (A) Solid-phase hybridization. Bait probes (light blue and black) complementary to the desired sequence are synthesized on a microarray. Fragmented genomic DNA is applied, and the desired fragments hybridize. The array is washed, and desired fragments are eluted. (B) Liquid-phase hybridization. Bait probes (light blue and black) complementary to the desired regions are synthesized, often using microarray technology. The probes are generally biotinylated (asterisk). The bait probes are mixed with fragmented genomic DNA, and the desired fragments hybridize to baits in solution. Streptavidin beads (black circles) are added to allow physical separation. The bead-bait complexes are washed, and desired DNA is eluted. (C) MIP. Single-stranded probes composed of a universal linker backbone (black line) and arms complementary to the sequence flanking desired regions (red and white) are synthesized, often using microarray or microfluidics technology. The probes are added to genomic DNA and hybridize in an inverted manner. A polymerase (yellow oval) fills in the gap between the two arms. A ligase (yellow star) seals the nick, resulting in a closed single-strand circle. Genomic DNA is digested with exonucleases, and the captured DNA is amplified using sequences in the universal backbone. (D) PEC. Biotinylated primers (red and white) are added to fragmented genomic DNA, where they hybridize to the desired sequence. A polymerase (yellow oval) extends the primer, creating a tighter interaction. Streptavidin beads (black circles) are added and are used to physically separate the desired DNA from the unwanted DNA. The desired DNA is then eluted.