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Beyond assembly: the increasing flexibility of single-molecule sequencing technology

Nature Reviews Genetics volume 24, pages 627–641 (2023)Cite this article

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Abstract

The maturation of high-throughput short-read sequencing technology over the past two decades has shaped the way genomes are studied. Recently, single-molecule, long-read sequencing has emerged as an essential tool in deciphering genome structure and function, including filling gaps in the human reference genome, measuring the epigenome and characterizing splicing variants in the transcriptome. With recent technological developments, these single-molecule technologies have moved beyond genome assembly and are being used in a variety of ways, including to selectively sequence specific loci with long reads, measure chromatin state and protein–DNA binding in order to investigate the dynamics of gene regulation, and rapidly determine copy number variation. These increasingly flexible uses of single-molecule technologies highlight a young and fast-moving part of the field that is leading to a more accessible era of nucleic acid sequencing.

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Fig. 1: Long-read targeted sequencing methods.
Fig. 2: Long-read, single-molecule methyltransferase footprinting methods can reveal heterogeneity and coordination of chromatin states.
Fig. 3: Applications of shorter-read sequencing (<5 kb) on single-molecule platforms.

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Acknowledgements

This work was supported by funding from the National Institutes of Health (grant no. R01 HG009190; National Human Genome Research Institute).

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Authors and Affiliations

  1. Department of Biomedical Engineering, Molecular Biology and Genetics, and Genetic Medicine, Johns Hopkins University, Baltimore, MD, USA

    Paul W. Hook & Winston Timp

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  1. Paul W. Hook
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  2. Winston Timp
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The authors contributed equally to all aspects of the article.

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Correspondence to Winston Timp.

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Competing interests

W.T. has two patents (8,748,091 and 8,394,584) licensed to ONT. W.T. has received travel funds to speak at symposia organized by ONT. P.H. declares no competing interests.

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Supplementary information

Glossary

Basecaller

An algorithm that converts the raw signal from nucleic acid sequencing into the bases that the signal represents.

Centromeres

The region of a chromosome where the kinetochore attaches during cell division, typically an extremely repetitive region.

Chemical bisulfite conversion

A method used to measure the DNA modifications 5-methylcytosine and 5-hydroxymethylcytosine. DNA is treated with sodium bisulfite, which converts unmodified cytosine to uracil, whereas modified cytosines are protected from conversion. Following conversion and PCR, unmodified cytosines are read as thymines when sequenced, whereas modified cytosines remain cytosines.

Chromatin immunoprecipitation followed by sequencing

(ChIP–seq). A method for directly measuring protein–DNA binding with antibody-mediated immunoprecipitation of protein–DNA complexes.

Cleavage under targets & release using nuclease

(CUT&RUN). A method for directly measuring protein–DNA binding with antibody-guided DNA digestion with a micrococcal nuclease.

Cleavage under targets and tagmentation

(CUT&Tag). A method for directly measuring protein–DNA binding with antibody-guided transposition and fragmentation (tagmentation) with Tn5 transposase.

Cycle dephasing

Mechanism of error that affects sequencing devices using polymerase colonies (polonies). This occurs when clonal molecules within the same cluster are not all elongated in a given extension step, diluting the sequencing signal during subsequent cycles as the molecules become out of phase. More molecules become ‘dephased’ with each additional sequencing cycle, leading to increasingly lower sequencing quality as different positions on the template contribute to the signal.

Dynamic time warping

An algorithm for measuring similarity between two time series. In this context it refers to matching experimental nanopore data to a modelled electrical signal from a reference DNA sequence to identify the correct sequence from a database.

Human Genome Project

An international effort launched in 1990 with the primary goal of assembling the human genome. The project was completed in 2003.

ONT Flongle flow cell

Low-throughput flow cell (<1 Gb) from Oxford Nanopore Technologies. This flow cell can be sequenced on MinION or GridION sequencing devices.

ONT MinION

Hand-held sequencing device from Oxford Nanopore Technologies that can perform sequencing with MinION or Flongle flow cells.

ONT MinION flow cell

Medium-throughput (2–20 Gb) flow cell from Oxford Nanopore Technologies. This flow cell can be sequenced on MinION or GridION sequencing devices.

ONT PromethION

High-throughput sequencing device from Oxford Nanopore Technologies that can perform sequencing with PromethION flow cells.

ONT PromethION flow cell

High-throughput (50–100+ Gb) flow cell from Oxford Nanopore Technologies. This flow cell can be sequenced on PromethION sequencing devices.

PacBio RS II

Sequencing device released by Pacific Biosciences in 2013 that can perform single-molecule, real-time sequencing.

PacBio Sequel II

Sequencing device released by Pacific Biosciences in 2019 that can perform single-molecule, real-time sequencing.

Sequencing depth

The number of reads that map to a given locus, also known as sequencing coverage. This is usually represented as an average, and a locus can refer to a single nucleotide, region(s) of interest, entire chromosome(s) or entire genomes. We would consider ‘high’ coverage or depth as >100× for most assays.

Telomeres

Repetitive regions at the end of chromosomes.

Tn5 transposase

A bacterial protein that facilitates the movement of DNA sequences through a ‘cut and paste’ mechanism. This protein has become a valuable molecular biology tool with its uses ranging from efficient library preparation to probing chromatin state.

Unique molecular identifiers

(UMIs). Short sequences of random nucleotides that tags an individual nucleic acid molecule. UMIs can be used to identify subsequently amplified fragments that arose from the same original molecule, mitigating bias introduced during PCR and allowing for more accurate quantification.

Whole-genome sequencing

A sequencing approach that attempts to obtain reads that map to all bases in the genome.

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Hook, P.W., Timp, W. Beyond assembly: the increasing flexibility of single-molecule sequencing technology. Nat Rev Genet 24, 627–641 (2023). https://doi.org/10.1038/s41576-023-00600-1

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