The chemistry of next-generation sequencing

Abstract

The first large genome fully sequenced by next-generation sequencing (NGS) was that of a bacteriophage using sequencing by synthesis (SBS) as a paradigm. SBS in NGS is underpinned by 'reversible-terminator chemistry'. To grow from proof of concept to being both affordable and practical, SBS needed to overcome a series of challenges, each of which required the invention of new chemistries. These included the design and synthesis of unnatural deoxynucleotide triphosphates (dNTPs), engineering a suitable polymerase, a new surface chemistry and an ingenious molecular solution to neutralize copying errors inherent to all polymerases. In this historical Perspective, we discuss how NGS was developed from Sanger sequencing, highlighting the chemistry behind this technology, which has impacted biology in unprecedented ways.

Figure 1.

Schematic illustration of Sanger Sequencing. DNA strands are separated based on size, which is defined by where into the chain a ddNTP is incorporated. This reveals the identity of the nucleobase at any position, as incorporation of a ddNTP terminates the growing chain at that position.

Figure 2.

Scheme of reversible terminator chemistry. Thymidine (blue) is functionalized with a fluorophore (red) linked at the C5 position with an azide (green)-containing cleavable linker. The ribose 3′-OH is masked by a cleavable azide (green)-containing cleavable protecting group. A Staudinger reaction using a water soluble triphenylphosphine converts the azide into hemiaminals, which upon hydrolysis releases the fluorophore and restores the free 3′-OH in a single step. This reaction product can be engaged in a new cycle of dNTP incorporation at 3′-OH.

Figure 3.

Schematic illustration of the active site within a DNA polymerase showing the close positioning of the 3′-OH (brown) of the elongating strand in line with the αP (cyan) of the incoming dNTP.

Figure 4.

Schematic of the workflow of NGS (Part 1). DNA libraries prepared by fragmenting genomic DNA into 200–300 base pairs to which adapters are attached. The library is amplified by PCR, and unique strands are hybridized on a solid support. Bridge amplification results in distinct colonies of identical strands.

Figure 5.

Schematic of the workflow of NGS (Part 2). The distinct colonies of identical strands are then subjected to SBS and imaged in a microfluidic flow-cell using the chemistry developed by Solexa.