Review Article | Published:

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

Nature Reviews Genetics volume 17, pages 333351 (2016) | Download Citation


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.

Key points

  • There are two major paradigms in next-generation sequencing (NGS) technology: short-read sequencing and long-read sequencing. Short-read sequencing approaches provide lower-cost, higher-accuracy data that are useful for population-level research and clinical variant discovery. By contrast, long-read approaches provide read lengths that are well suited for de novo genome assembly applications and full-length isoform sequencing.

  • NGS technologies have been evolving over the past 10 years, leading to substantial improvements in quality and yield; however, certain approaches have proven to be more effective and adaptable than others.

  • Recent improvements in chemistry, costs, throughput and accessibility are driving the emergence of new, varied technologies to address applications that were not previously possible. These include integrated long-read and short-read sequencing studies, routine clinical DNA sequencing, real-time pathogen DNA monitoring and massive population-level projects.

  • Although massive strides are being made in this technology, several notable limitations remain. The time required to sequence and analyse data limits the use of NGS in clinical applications in which time is an important factor; the costs and error rates of long-read sequencing make it prohibitive for routine use, and ethical considerations can limit the public and private use of genetic data.

  • We can expect increasing democratization and options for NGS in the future. Many new instruments with varied chemistries and applications are being released or being developed.

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Author information


  1. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.

    • Sara Goodwin
    •  & W. Richard McCombie
  2. Department of Biochemistry and Molecular Medicine; and the Comprehensive Cancer Center, University of California, Davis, California 95817, USA.

    • John D. McPherson


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Competing interests

W.R.M. and J.D.M. have participated in Illumina sponsored meetings over the past four years and received travel reimbursement and honoraria for presenting at these events. Illumina had no role in decisions relating to the study/work to be published, data collection and analysis of data and the decision to publish. W.R.M. and J.D.M. have participated in Pacific Biosciences sponsored meetings over the past three years and received travel reimbursement for presenting at these events. S.H.G. has participated in Oxford Nanopore Technologies sponsored meetings and received travel reimbursement for presenting at these events.

Corresponding author

Correspondence to W. Richard McCombie.



The sequence of bases from a single molecule of DNA.

Sanger sequencing

An approach in which dye-labelled normal deoxynucleotides (dNTPs) and dideoxy-modified dNTPs are mixed. A standard PCR reaction is carried out and, as elongation occurs, some strands incorporate a dideoxy-dNTP, thus terminating elongation. The strands are then separated on a gel and the terminal base label of each strand is identified by laser excitation and spectral emission analysis.


A DNA fragment to be sequenced. The DNA is typically ligated to one or more adapter sequences where DNA sequencing will be initiated.


The process of breaking large DNA fragments into smaller fragments. This can be achieved mechanically (by passing the DNA through a narrow passage), by sonication or enzymatically.


Groups of DNA templates in close spatial proximity, generated either though bead-based amplification or by solid-phase amplification. Bead-based approaches rely on emulsions to maintain template isolation during amplification. Solid-phase approaches rely on the template-to-bound-adapter ratio to probabilistically bind template molecules at a sufficient distance from each other.

Flow cells

Disposable parts of a next-generation sequencing routine. Template DNA is immobilized within the flow cell where fluid reagents can be streamed into the cell and flushed away.

Rolling circle amplification

(RCA). A method of DNA amplification using a circular template. Briefly, DNA polymerase binds to a primed section of a circular DNA template. As the polymerase traverses the template, a new strand is synthesized. When the polymerase completes a full circle and encounters the double-stranded DNA (dsDNA) template, it displaces the template without degradation, thus creating a long ssDNA fragment composed of many copies of the template sequence.

One-base-encoded probes

Oligonucleotides that contain a single interrogation base in a known position. The base corresponds to a fluorescent label on each probe. The remaining bases are either degenerate (any of the four bases) or universal (unnatural bases with nonspecific hybridization), allowing the probe to interact with many different possible template sequences.

Two-base-encoded probes

Oligonucleotides that contain two adjacent interrogation bases in a known position. The bases correspond to a fluorescent label on each probe. The remaining bases are either degenerate (any of the four bases) or universal (unnatural bases with nonspecific hybridization) allowing the probe to interact with many different possible template sequences.


A system exclusively used by SOLiD. When a two-base-encoded probe is used, the bound label corresponds to two bases rather than one. Thus, the signal derived from a SOLiD run is in a series of colours that represent overlapping dinucleotides, rather than each colour being directly correlated to a single base. A reference-based alignment is the most efficient way to translate colour-space into base-space. For example, in the sequence ATGT the first probe will match AT, the second will match TG and the third GT. If the AT is known, then the subsequent colour order is uniquely solved as TG and GT, leading to a readout of ATGT. Final sequence deconvolution of colour-space is achieved with the knowledge of the second base identity in one round and the colour of the subsequent round in which the ligation is offset by one nucleotide, allowing for the identification of the next base.


A system used by most next-generation sequencing platforms. When a one-base-encoded probe or a sequencing-by-synthesis approach is used, each signal is correctly correlated to a base.

Whole-genome sequencing

(WGS). Sequencing of the entire genome without using methods for sequence selection.

Two-fluorophore system

A system in which bases are discriminated by labelling Cs and Ts with a red or green fluorophore, respectively. Each A base is labelled with either a red or green fluorophore, but the two populations are mixed. During base discrimination, clusters that are either red or green are called either C or T, whereas clusters with a red and green mixed signal are called A. The G base is unlabelled, thus any cluster without a fluorophore signal is called G.


A sequence run of identical bases.

Charge-coupled device

(CCD). A device composed of an integrated circuit that forms light-sensitive elements: pixels. When a photon interacts with the device, the light generates a charge that can be interpreted by an electronic device.

Integrated complementary metal-oxide-semiconductor

(CMOS). An integrated circuit design that is printed on a microchip that contains different types of semiconductor transistors to create a circuit that both uses very little power and is resistant to high levels of electronic noise.

Ion-sensitive field-effect transistor

(ISFET). A type of transistor that is sensitive to changes in ion concentration.

Single-end and paired-end sequencing

In single-end sequencing, a DNA template is sequenced only in one direction. In paired-end sequencing, a DNA template is sequenced from both sides; the forward and reverse reads may or may not overlap. A deviation in the expected genome alignment between two ends of a paired-end read can indicate astructural variation.

Structural variant

A variation larger than single-nucleotide polymorphisms (SNPs). This can include the insertion or deletion of blocks of DNA, inversions or translocations of DNA segments, and copy-number differences.


(Chromatin immunoprecipitation followed by sequencing). A method used to analyse protein interactions with DNA by combining ChIP with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins.


(Assay for transposase-accessible chromatin with high-throughput sequencing). A method that uses the activity of a hyperactive transposase to cleave exposed DNA and add sequencing adapters. Regions that cannot be sequenced are inferred to be chromatin interacting.

RNA sequencing

(RNA-seq). A method of sequencing cDNA derived from RNA. This approach can be used to sequence both coding and non-coding RNA.

Real-time sequencing

A sequencing strategy used in the Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) platforms. In these approaches there is no pause after the detection of a base or series of bases, thus the sequence is derived in real-time.


A series of known bases added to a template molecule either through ligation or amplification. After sequencing, these barcodes can be used to identify which sample a particular read is derived from.

Zero-mode waveguides

(ZMW). Nanostructure devices used in the Pacific Biosciences (PacBio) platform. Each ZMW well (also called a waveguide) is several nanometres in diameter and is anchored to a glass substrate. The size of each well does not allow for light propagation, thus the fluorophores bound to bases can only be visualized through the glass substrate in the bottom-most portion of the well, a volume in the zeptolitre range.

Read of insert

The highest-quality single sequence for an insert, regardless of the number of passes.

Consensus sequence

In next-generation sequencing (NGS) routines that allow multiple overlapping reads from a single molecule of DNA, all related reads are aligned to each other and the most likely base at each position is determined. This process helps to overcome high, single-pass error rates. A high-quality consensus sequence derived from the circular template from Pacific Biosciences (PacBio) is called a circular consensus sequence (CCS).

Squiggle space

A system exclusively used by Oxford Nanopore Technologies (ONT). As DNA translocates through the pore, a shift in voltage occurs that is directly correlated to a k-mer within the pore. Thus, the signal derived from a nanopore run is a continuous series of voltage shifts (squiggles) that represent a series of overlapping k-mers.


A substring within a sequence of bases of some (k) length. Currently, k-mer sizes of Oxford Nanopore Technologies (ONT) range from 3 to 6 bases.

1D and 2D reads

Oxford Nanopore Technologies (ONT) sequencing allows for both the full forward and full reverse strand of a double-stranded DNA (dsDNA) molecule to be sequenced and associated. A 1D read is the sequence of DNA bases derived from either the forward or reverse DNA strand. A 2D read is a consensus sequence derived from both the forward and the reverse reads.

BAC-by-BAC sequencing

A sequencing method where a physical map is generated from overlapping bacterial artificial chromosome (BAC) clones tiled across a chromosome. Each BAC is then fragmented and sequenced. The sequenced fragments are aligned with the knowledge of the originating BAC.

Linked reads

Reads derived from the 10X Genomics synthetic long-read platform. These are discontinuous reads each sharing the same barcode, thus they are derived from the same original long molecule.

Read cloud

The means by which the 10X Genomics platform determines a synthetic long read. Discontinuous linked reads from the same genomic region are aligned to each other. No single linked read contains the entire long sequence; however, when they are stacked, full coverage is achieved.

Polymerase reads

Contiguous sequences of nucleotides incorporated by the DNA polymerase while reading a template. These reads include sequences from adapters and can represent sequences from multiple passes around a circular template.


The single-molecule real-time (SMRT) sequencing approach from Pacific Biosciences (PacBio) enables a single molecule of DNA to be sequenced multiple times. A single pass is one single iteration through a molecule.


The sequences derived from a single pass as a polymerase traverses a DNA molecule multiple times. A subread is trimmed to exclude any adapter sequence.

Whole-exome and targeted sequencing

Sequencing of only exons or other selected regions. A system of capture or amplification is used to isolate or enrich for only exons or target regions. This is done by designing probes or primers for the regions of interest.

Genome phasing

A method to identify which chromosome a DNA sequence is derived from. By examining polymorphisms, the chromosome of origin can be inferred by matching the reads that share the same variation.

Family studies

A study design in which many members of a family across several generations are sequenced. These studies are used to understand how phenotypes manifest within a particular genotype background.

Helicos Genetic Analysis System

A sequencing technology based on single nucleotide addition. Each nucleotide contains a 'virtual terminator' that prevents the incorporation of multiple nucleotides per cycle.

Fluorescence resonance energy transfer

(FRET; also known as Förster resonance energy transfer). A system in which energy can be transferred from one light-sensitive molecule to another. When the two molecules are in close proximity (≤30 nm), energy transferred between the two molecules modulates the intensity of a fluorescence signal.

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