Coming of age: ten years of next-generation sequencing technologies

Abstract

Since the completion of the human genome project in 2003, extraordinary progress has been made in genome sequencing technologies, which has led to a decreased cost per megabase and an increase in the number and diversity of sequenced genomes. An astonishing complexity of genome architecture has been revealed, bringing these sequencing technologies to even greater advancements. Some approaches maximize the number of bases sequenced in the least amount of time, generating a wealth of data that can be used to understand increasingly complex phenotypes. Alternatively, other approaches now aim to sequence longer contiguous pieces of DNA, which are essential for resolving structurally complex regions. These and other strategies are providing researchers and clinicians a variety of tools to probe genomes in greater depth, leading to an enhanced understanding of how genome sequence variants underlie phenotype and disease.

Figure 1 |. Template amplification strategies.

a | In emulsion PCR, fragmented DNA templates are ligated to adapter sequences and are captured in an aqueous droplet (micelle) along with a bead covered with complementary adapters, deoxynucleotides (dNTPs), primers and DNA polymerase. PCR is carried out within the micelle, covering each bead with thousands of copies of the same DNA sequence. b | In solid-phase bridge amplification, fragmented DNA is ligated to adapter sequences and bound to a primer immobilized on a solid support, such as a patterned flow cell. The free end can interact with other nearby primers, forming a bridge structure. PCR is used to create a second strand from the immobilized primers, and unbound DNA is removed. c | In solid-phase template walking, fragmented DNA is ligated to adapters and bound to a complementary primer attached to a solid support. PCR is used to generate a second strand. The now double-stranded template is partially denatured, allowing the free end of the original template to drift and bind to another nearby primer sequence. Reverse primers are used to initiate strand displacement to generate additional free templates, each of which can bind to a new primer. d | In DNA nanoball generation, DNA is fragmented and ligated to the first of four adapter sequences. The template is amplified, circularized and cleaved with a type II endonuclease. A second set of adapters is added, followed by amplification, circularization and cleavage. This process is repeated for the remaining two adapters. The final product is a circular template with four adapters, each separated by a template sequence. Library molecules undergo a rolling circle amplification step, generating a large mass of concatamers called DNA nanoballs, which are then deposited on a flow cell. Parts a and b are adapted from REF. , Nature Publishing Group.

Figure 2 |. Sequencing by ligation methods.

a | SOLiD sequencing. Following cluster generation or bead deposition onto a slide, fragments are sequenced by ligation, in which a fluorophore-labelled two-base-encoded probe, which is composed of known nucleotides in the first and second positions (dark blue), followed by degenerate or universal bases (pink), is added to the DNA library. The two-base probe is ligated onto an anchor (light purple) that is complementary to an adapter (red), and the slide is imaged to identify the first two bases in each fragment. Unextended strands are capped by unlabelled probes or phosphatase to maintain cycle synchronization. Finally, the terminal degenerate bases and the fluorophore are cleaved off the probe, leaving a 5 bp extended fragment. The process is repeated ten times until two out of every five bases are identified. At this point, the entire strand is reset by removing all of the ligated probes and the process of probe binding, ligation, imaging and cleavage is repeated four times, each with an n + 1, n + 2, n + 3 or n + 4 offset anchor. b | Complete Genomics. DNA is sequenced using the combinatorial probe–anchor ligation (cPAL) approach. After DNA nanoball deposition, an anchor complementary to one of four adapter sequences and a fluorophore-labelled probe are bound to each nanoball. The probe is degenerate at all but the first position. The anchor and probe are then ligated into position and imaged to identify the first base on either the 3′ or the 5′ side of the anchor. Next, the probe–anchor complex is removed and the process begins again with the same anchor but a different probe with the known base at the n + 1 position. This is repeated until five bases from the 3′ end of the anchor and five bases from the 5′ end of the anchor are identified. Another round of hybridization occurs, this time using anchors with a five-base offset identifying an additional five bases on either side of the anchor. Finally, this whole process is repeated for each of the remaining three adapter sequences in the nanoball, generating 100 bp paired-end reads.

Figure 3 |. Sequencing by synthesis: cyclic reversible termination approaches.

a | Illumina. After solid-phase template enrichment, a mixture of primers, DNA polymerase and modified nucleotides are added to the flow cell. Each nucleotide is blocked by a 3′-O-azidomethyl group and is labelled with a base-specific, cleavable fluorophore (F). During each cycle, fragments in each cluster will incorporate just one nucleotide as the blocked 3′ group prevents additional incorporations. After base incorporation, unincorporated bases are washed away and the slide is imaged by total internal reflection fluorescence (TIRF) microscopy using either two or four laser channels; the colour (or the lack or mixing of colours in the two-channel system used by NextSeq) identifies which base was incorporated in each cluster. The dye is then cleaved and the 3′-OH is regenerated with the reducing agent tris(2-carboxyethyl)phosphine (TCEP). The cycle of nucleotide addition, elongation and cleavage can then begin again. b | Qiagen. After bead-based template enrichment, a mixture of primers, DNA polymerase and modified nucleotides are added to the flow cell. Each nucleotide is blocked by a 3′-O-allyl group and some of the bases are labelled with a base-specific, cleavable fluorophore. After base incorporation, unincorporated bases are washed away and the slide is imaged by TIRF using four laser channels. The dye is then cleaved and the 3′-OH is regenerated with the reducing agent mixture of palladium and P(PhSO₃Na)₃ (TPPTS).

Figure 4 |. Sequencing by synthesis: single-nucleotide addition approaches.

a | 454 pyrosequencing. After bead-based template enrichment, the beads are arrayed onto a microtitre plate along with primers and different beads that contain an enzyme cocktail. During the first cycle, a single nucleotide species is added to the plate and each complementary base is incorporated into a newly synthesized strand by a DNA polymerase. The by-product of this reaction is a pyrophosphate molecule (PP_i). The PP_i molecule, along with ATP sulfurylase, transforms adenosine 5′ phosphosulfate (APS) into ATP. ATP, in turn, is a cofactor for the conversion of luciferin to oxyluciferin by luciferase, for which the by-product is light. Finally, apyrase is used to degrade any unincorporated bases and the next base is added to the wells. Each burst of light, detected by a charge-coupled device (CCD) camera, can be attributed to the incorporation of one or more bases at a particular bead. b | Ion Torrent. After bead-based template enrichment, beads are carefully arrayed into a microtitre plate where one bead occupies a single reaction well. Nucleotide species are added to the wells one at a time and a standard elongation reaction is performed. As each base is incorporated, a single H⁺ ion is generated as a by-product. The H⁺ release results in a 0.02 unit change in pH, detected by an integrated complementary metal-oxide semiconductor (CMOS) and an ion-sensitive field-effect transistor (ISFET) device. After the introduction of a single nucleotide species, the unincorporated bases are washed away and the next is added. Part a is adapted from REF. , Nature Publishing Group.

Figure 5 |. Real-time and synthetic long-read sequencing approaches.

A | Real-time long-read sequencing platforms. Aa | Single-molecule real-time (SMRT) sequencing from Pacific Biosciences (PacBio). Template fragments are processed and ligated to hairpin adapters at each end, resulting in a circular DNA molecule with constant single-stranded DNA (ssDNA) regions at each end with the double-stranded DNA (dsDNA) template in the middle. The resulting ‘SMRTbell’ template undergoes a size-selection protocol in which fragments that are too large or too small are removed to ensure efficient sequencing. Primers and an efficient φ29 DNA polymerase are attached to the ssDNA regions of the SMRTbell. The prepared library is then added to the zero-mode waveguide (ZMW) SMRT cell, where sequencing can take place. To visualize sequencing, a mixture of labelled nucleotides is added; as the polymerase-bound DNA library sits in one of the wells in the SMRT cell, the polymerase incorporates a fluorophore-labelled nucleotide into an elongating DNA strand. During incorporation, the nucleotide momentarily pauses through the activity of the polymerase at the bottom of the ZMW, which is being monitored by a camera. Ab | Oxford Nanopore Technologies (ONT). DNA is initially fragmented to 8–10 kb. Two different adapters, a leader and a hairpin, are ligated to either end of the fragmented dsDNA. Currently, there is no method to direct the adapters to a particular end of the DNA molecule, so there are three possible library conformations: leader–leader, leader–hairpin and hairpin–hairpin. The leader adapter is a double-stranded adapter containing a sequence required to direct the DNA into the pore and a tether sequence to help direct the DNA to the membrane surface. Without this leader adapter, there is minimal interaction of the DNA with the pore, which prevents any hairpin–hairpin fragments from being sequenced. The ideal library conformation is the leader–hairpin. In this conformation the leader sequence directs the DNA fragment to the pore with current passing through. As the DNA translocates through the pore, a characteristic shift in voltage through the pore is observed. Various parameters, including the magnitude and duration of the shift, are recorded and can be interpreted as a particular k-mer sequence. As the next base passes into the pore, a new k-mer modulates the voltage and is identified. At the hairpin, the DNA continues to be translocated through the pore adapter and onto the complement strand. This allows the forward and reverse strands to be used to create a consensus sequence called a ‘2D’ read. B | Synthetic long-read sequencing platforms. Ba | Illumina. Genomic DNA templates are fragmented to 8–10 kb pieces. They are then partitioned into a microtitre plate such that there are around 3,000 templates in a single well. Within the plate, each fragment is sheared to around 350 bp and barcoded with a single barcode per well. The DNA can then be pooled and sent through standard short-read pipelines. Bb | 10X Genomics’ emulsion-based sequencing. With as little as 1 ng of starting material, the GemCode can partition arbitrarily large DNA fragments, up to ~100 kb, into micelles (also called ‘GEMs’) along with gel beads containing adapter and barcode sequences. The GEMs typically contain ~0.3× copies of the genome and 1 unique barcode out of 750,000. Within each GEM, the gel bead dissolves and smaller fragments of DNA are amplified from the original large fragments, each with a barcode identifying the source GEM. After sequencing, the reads are aligned and linked together to form a series of anchored fragments across a span of ~50 kb. Unlike the Illumina system, this approach does not attempt to get full end-to-end coverage of a single DNA fragment. Instead, the reads from a single GEM are dispersed across the original DNA fragment and the cumulative coverage is derived from multiple GEMs with dispersed — but linked — reads. Part Aa is adapted from REF. , Nature Publishing Group. Part Ba is adapted from REF. .