High-throughput sequencing technologies

Abstract

The human genome sequence has profoundly altered our understanding of biology, human diversity, and disease. The path from the first draft sequence to our nascent era of personal genomes and genomic medicine has been made possible only because of the extraordinary advancements in DNA sequencing technologies over the past 10 years. Here, we discuss commonly used high-throughput sequencing platforms, the growing array of sequencing assays developed around them, as well as the challenges facing current sequencing platforms and their clinical application.

Figure 1. Timeline and comparison of commercial HTS instruments

Plot of commercial release dates versus machine outputs per run are shown. For the MinION, outputs from an 18 hour run were used (Ashton et al., 2014). Numbers inside data points denote current read lengths. Sequencing platforms are color-coded.

Figure 2. Clonal amplification-based sequencing platforms

(A) Illumina’s four-color reversible termination sequencing method. DNA templates are first clonally amplified on the surface of a glass flow cell. Sequencing occurs via successive rounds of base incorporation, washing and imaging. A cleavage step after image acquisition removes the fluorescent dye and regenerates the 3´OH for the next cycle. Analysis of four-color images is used to determine base composition. (B) Ion Torrent’s semiconductor sequencing method. Emulsion-PCR is used to clonally amplify DNA templates on the surface of beads, which are subsequently placed into microwells. pH changes induced by the release of hydrogen ions during DNA extension are detected by a sensor positioned at the bottom of the microwell. These pH changes are converted into a voltage signal, which is proportional to the number of nucleotides added by the polymerase.

Figure 3. Single molecule sequencing platforms

(A) Pacific Bioscience’s SMRT sequencing. A single polymerase is positioned at the bottom of a zero-mode waveguide (ZMW). Phosphate-labeled versions of all 4 nucleotides are present, allowing continuous polymerization of a DNA template. Base incorporation increases the residence time of the nucleotide in the ZMW, resulting in a detectable fluorescent signal that is captured in a video. (B) Oxford Nanopore’s sequencing strategy. DNA templates are ligated with two adapters. The first adapter is bound with a motor enzyme as well as a tether, whereas the second adapter is a hairpin oligo that is bound by the HP motor protein. Changes in current that are induced as the nucleotides pass through the pore are used to discriminate bases. The library design allows sequencing of both strands of DNA from a single molecule (2-direction reads).

Figure 4. Broad overview of HTS applications

Publication date of a founding article describing a method versus the number of citations that the article received. Methods are colored by category, and the size of the data point is proportional to publication rate (citations/months). The inset indicates the color key as well the proportion of methods in each group. For clarity, Seq has omitted from the labels.