Figure 1
Hybrid capture enriched next generation sequencing. Although next generation sequencing encompasses a variety of technologies, each relies on massive parallelization of sequencing to achieve enormous throughput. In this example, Illumina sequencing is depicted. (a) Genomic DNA is sheared into small pieces (typically 300–500 bp) by sonication. (b) Sequencing adapters and sequencing indexes, the latter allowing for the identification of individual samples in pooled multiplexed data, are ligated to the sheared genomic DNA. (c) The prepared DNA (now called a library) is captured using biotinnylated cRNA oligomers specific for the region of interest. Following hybridization, this enriched DNA is eluted. (d) The enriched DNA libraries from multiple samples (each with unique index tags) are then loaded into a ‘flow cell’ containing immobilized oligomers with sequences complementary to the ligated library adapters. The library DNA then binds the surface and undergoes ‘bridge amplification’ to produce small colonies containing the amplified DNA library sequence. (e) DNA in the colonies is then sequenced using fluorescent, reversibly blocked nucleotides. Each nucleotide is labeled with a unique fluorophore, and, following incorporation of the complementary labeled base, each colony is scanned by a laser to determine the sequence of the last incorporated base. This process is performed in parallel over millions of colonies and repeated for each base, resulting in reads ranging from 36 to 150 bp depending on the chemistry and instrument used.
