Sequencing Techniques

Abstract

For decades, Sanger’s chain termination method was synonym to DNA sequencing. Two new generations of sequencing platforms were launched since the mid-2000s which transformed the field of evolutionary genomics. The first commercially available next-generation sequencing technique was 454 pyrosequencing. This technique is based on emulsion PCR, and sequencing is achieved by detecting light signals triggered by cyclically flowed nucleotides. Illumina’s reverse terminator sequencing is today the dominant technique used for genome sequencing. DNA molecules attached to flow cells and clonally amplified via bridge amplification are read out step by step adding fluorescent-labelled nucleotides. The output of Illumina sequencing runs is currently the highest while being most cost-efficient at the same time. A technique that tries to rival this position is Ion Torrent’s semiconductor sequencing. Without any optical devices, changes in the ion concentration due to the release of protons from nucleotides which are incorporated into a DNA strand by polymerase are measured. The read length of all these techniques is limited to 200–1000 bps. A third generation of sequencing techniques eliminated all PCR steps during library preparation, therefore enabling single-molecule sequencing. PacBio’s single-molecule real-time sequencing records the emission from phospho-linked nucleotides while incorporated into DNA strands. Average read lengths of ~8 kbp are attained by these sequencers, and epigenetically modified nucleotides can be detected simultaneously. Superfast nanopore sequencing as realized in Oxford Nanopore Technologies MinION allows the detection of nucleotides while being ratcheted through a biological pore enabling real-time genome sequencing with easy handling, opening up new possibilities. Even still error prone, read lengths are in theory only limited by chromosome length with nanopore sequencing.