The sequence of sequencers: The history of sequencing DNA The sequence of sequencers: The history of sequencing DNA
Highlights
We review the drastic changes to DNA sequencing technology over the last 50 years.
First-generation methods enabled sequencing of clonal DNA populations.
The second-generation massively increased throughput by parallelizing many reactions.
Third-generation methods allow direct sequencing of single DNA molecules.
Abstract
whole genome sequencing. This article traverses those years, iterating through the different generations of sequencing technology, highlighting some of the key discoveries, researchers, and sequences along the way.
Keywords
1. Introduction
“... [A] knowledge of sequences could contribute much to our understanding of living matter.”
Frederick Sanger [1]
sequence DNA, and the characteristics that define each generation of methodologies for doing so.
2. First-generation DNA sequencing
[4]. New tactics needed to be developed.
[16].
[21]. However the actual determination of bases was still restricted to short stretches of DNA, and still typically involved a considerable amount of analytical chemistry and fractionation procedures.
DNA sequencing.
electrophoresis on a high-resolution polyacrylamide gel: sequences are then inferred by reading ‘up’ the gel, as the shorter DNA fragments migrate fastest. In Sanger sequencing (left) the sequence is inferred by finding the lane in which the band is present for a given site, as the 3′ terminating labelled ddNTP corresponds to the base at that position. Maxam–Gilbert sequencing requires a small additional logical step: Ts and As can be directly inferred from a band in the pyrimidine or purine lanes respectively, while G and C are indicated by the presence of dual bands in the G and A + G lanes, or C and C + T lanes respectively.
Sanger sequencing – to become the most common technology used to sequence DNA for years to come.
[34] which were used to sequence the genomes of increasingly complex species.
[46].
3. Second-generation DNA sequencing
[51]. Pyrosequencing was later licensed to 454 Life Sciences, a biotechnology company founded by Jonathan Rothburg, where it evolved into the first major successful commercial ‘next-generation sequencing’ (NGS) technology.
[57].
[62].
nucleotides at the ends of a proceeding extension reaction, requiring cycle-by-cycle measurements and removal of terminators.
[70].
[72] and thus can probably considered to have made the greatest contribution to the second-generation of DNA sequencers.
4. Third-generation DNA sequencing
DNA amplification shared by all previous technologies.
[80] other companies took up the third-generation baton.
[81].
polymerisation at the base of the ZMW provide real-time bursts of fluorescent signal, without undue interference from other labelled dNTPs in solution. (b): Nanopore DNA sequencing as employed in ONT's MinION sequencer. Double stranded DNA gets denatured by a processive enzyme (†) which ratchets one of the strands through a biological nanopore (‡) embedded in a synthetic membrane, across which a voltage is applied. As the ssDNA passes through the nanopore the different bases prevent ionic flow in a distinctive manner, allowing the sequence of the molecule to be inferred by monitoring the current at each channel.
[99]. Nanopore sequencers could therefore revolutionize not just the composition of the data that can be produced, but where and when it can be produced, and by whom.
5. Conclusions
nucleotides in length. Over the years, innovations in sequencing protocols, molecular biology and automation increased the technological capabilities of sequencing while decreasing the cost, allowing the reading of DNA hundreds of basepairs in length, massively parallelized to produce gigabases of data in one run. Researchers moved from the lab to the computer, from pouring over gels to running code. Genomes were decoded, papers published, companies started – and often later dissolved – with repositories of DNA sequence data growing all the while. Therefore DNA sequencing – in many respects a relatively recent and forward-focussed research discipline – has a rich history. An understanding of this history can provide appreciation of current methodologies and provide new insights for future ones, as lessons learnt in the previous generation inform the progress of the next.