The sequence of sequencers: The history of sequencing DNA

Abstract

Determining the order of nucleic acid residues in biological samples is an integral component of a wide variety of research applications. Over the last fifty years large numbers of researchers have applied themselves to the production of techniques and technologies to facilitate this feat, sequencing DNA and RNA molecules. This time-scale has witnessed tremendous changes, moving from sequencing short oligonucleotides to millions of bases, from struggling towards the deduction of the coding sequence of a single gene to rapid and widely available 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: DNA; History; RNA; Sequencer; Sequencing.

Fig. 1

First-generation DNA sequencing technologies. Example DNA to be sequenced (a) is illustrated undergoing either Sanger (b) or Maxam–Gilbert (c) sequencing. (b): Sanger's ‘chain-termination’ sequencing. Radio- or fluorescently-labelled ddNTP nucleotides of a given type - which once incorporated, prevent further extension - are included in DNA polymerisation reactions at low concentrations (primed off a 5′ sequence, not shown). Therefore in each of the four reactions, sequence fragments are generated with 3′ truncations as a ddNTP is randomly incorporated at a particular instance of that base (underlined 3′ terminal characters). (c): Maxam and Gilbert's ‘chemical sequencing’ method. DNA must first be labelled, typically by inclusion of radioactive P ³² in its 5′ phosphate moiety (shown here by Ⓟ). Different chemical treatments are then used to selectively remove the base from a small proportion of DNA sites. Hydrazine removes bases from pyrimidines (cytosine and thymine), while hydrazine in the presence of high salt concentrations can only remove those from cytosine. Acid can then be used to remove the bases from purines (adenine and guanine), with dimethyl sulfate being used to attack guanines (although adenine will also be affected to a much lesser extent). Piperidine is then used to cleave the phophodiester backbone at the abasic site, yielding fragments of variable length. (d): Fragments generated from either methodology can then be visualized via 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.

Fig. 2

Second-generation DNA sequencing parallelized amplification. (a): DNA molecules being clonally amplified in an emulsion PCR (emPCR). Adapter ligation and PCR produces DNA libraries with appropriate 5′ and 3′ ends, which can then be made single stranded and immobilized onto individual suitably oligonucleotide-tagged microbeads. Bead-DNA conjugates can then be emulsified using aqueous amplification reagents in oil, ideally producing emulsion droplets containing only one bead (illustrated in the two leftmost droplets, with different molecules indicated in different colours). Clonal amplification then occurs during the emPCR as each template DNA is physically separate from all others, with daughter molecules remaining bound to the microbeads. This is the conceptual basis underlying sequencing in 454, Ion Torrent and polony sequencing protocols. (b): Bridge amplification to produce clusters of clonal DNA populations in a planar solid-phase PCR reaction, as occurs in Solexa/Illumina sequencing. Single-stranded DNA with terminating sequences complementary to the two lawn-oligos will anneal when washed over the flow-cell, and during isothermal PCR will replicate in a confined area, bending over to prime at neighbouring sites, producing a local cluster of identical molecules. (c) and (d) demonstrate how these two different forms of clonally-amplified sequences can then be read in a highly parallelized manner: emPCR-produced microbeads can be washed over a picotiter plate, containing wells large enough to fit only one bead (c). DNA polymerase can then be added to the wells, and each nucleotide can be washed over in turn, and dNTP incorporation monitored (e.g. via pyrophosphate or hydrogen ion release). Flow-cell bound clusters produced via bridge amplification (d) can be visualized by detecting fluorescent reversible-terminator nucleotides at the ends of a proceeding extension reaction, requiring cycle-by-cycle measurements and removal of terminators.

Fig. 3

Third-generation DNA sequencing nucleotide detection. (a): Nucleotide detection in a zero-mode waveguide (ZMW), as featured in PacBio sequencers. DNA polymerase molecules are attached to the bottom of each ZMW (*), and target DNA and fluorescent nucleotides are added. As the diameter is narrower than the excitation light's wavelength, illumination rapidly decays travelling up the ZMW: nucleotides being incorporated during 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.