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Long-read human genome sequencing and its applications

Abstract

Over the past decade, long-read, single-molecule DNA sequencing technologies have emerged as powerful players in genomics. With the ability to generate reads tens to thousands of kilobases in length with an accuracy approaching that of short-read sequencing technologies, these platforms have proven their ability to resolve some of the most challenging regions of the human genome, detect previously inaccessible structural variants and generate some of the first telomere-to-telomere assemblies of whole chromosomes. Long-read sequencing technologies will soon permit the routine assembly of diploid genomes, which will revolutionize genomics by revealing the full spectrum of human genetic variation, resolving some of the missing heritability and leading to the discovery of novel mechanisms of disease.

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Fig. 1: Overview of short-read sequencing technologies.
Fig. 2: Overview of long-read sequencing technologies.
Fig. 3: PacBio and ONT long-read data types.
Fig. 4: Long-read data improve genome assembly.
Fig. 5: Long-read data provide insights into the biological relevance of structural variation and human evolution and diversity.
Fig. 6: Long-read platforms can be used to sequence RNA and detect nucleic acid modifications.

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Acknowledgements

The authors thank M. J. Chaisson and D. Porubsky for assistance with the figures, K. Munson for technical assistance and commentarial insight and T. Brown for assistance in editing the manuscript. This work was supported, in part, by grants from the US National Institutes of Health (HG010169 to E.E.E.) and the US National Institute of General Medical Sciences (1F32GM134558-01 to G.A.L.). M.R.V. was supported by a US National Library of Medicine Big Data Training Grant for Genomics and Neuroscience (5T32LM012419-04). E.E.E. is an investigator of the Howard Hughes Medical Institute.

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The authors contributed equally to all aspects of the article.

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Correspondence to Evan E. Eichler.

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E.E.E. is on the scientific advisory board of DNAnexus Inc.

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Nature Reviews Genetics thanks M. Schatz and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

All of Us: https://allofus.nih.gov/

Arrow: https://github.com/PacificBiosciences/GenomicConsensus

Loman Labs: https://lab.loman.net/2017/03/09/ultrareads-for-nanopore/

Medaka: https://github.com/nanoporetech/medaka

Nanopolish: https://github.com/jts/nanopolish

Pacific Biosciences: does speed impact quality and yield?: https://github.com/PacificBiosciences/ccs#does-speed-impact-quality-and-yield

Supplementary Information

Glossary

Next-generation sequencing

A sequencing method in which an entire genome is sequenced from fragmented DNA, producing short (less than 300 bp) sequencing reads at high speed and low cost.

Sequence-by-synthesis

A sequencing technology used primarily by Illumina, in which a DNA polymerase synthesizes a strand of DNA complementary to a template by incorporating a fluorescently labelled deoxynucleoside triphosphate that is imaged to identify the base and then cleaved before the process is repeated to determine the order and identity of each base in the DNA strand.

Single-nucleotide variants

Instances in which a single base within a read or genome differs from the base found at the same position in other individuals or populations.

Copy number variants

Instances in which a sequence of bases within a genome differs in the number of copies among individuals or populations.

Indels

Insertions or deletions of bases in the genome of an organism.

Structural variant

A genetic variant greater than 50 bp in length that includes insertions, deletions, inversions or translocations of DNA segments, and copy number differences.

Segmental duplications

Blocks of DNA that are greater than 1 kb in length, occur at more than one site within a genome and share greater than 90% sequence identity.

Linked-read sequencing

A synthetic long-read DNA sequencing method wherein short-read sequencing is applied to long DNA molecules to ‘link’ reads together from the same original long molecule.

Long-read sequencing

A sequencing method used by Pacific Biosciences and Oxford Nanopore Technologies, wherein native DNA or RNA molecules are sequenced in real time, often without the need for amplification, producing reads more than 10 kb in length.

Contigs

Continuous (or ‘contiguous’) sequences of DNA generated by assembling overlapping sequencing reads.

Single-molecule, real-time (SMRT) sequencing

A DNA sequencing method used by Pacific Biosciences wherein the sequence of a single DNA molecule is derived in real time, with no pause after the detection of the bases.

SMRTbell

A double-stranded DNA template used in Pacific Biosciences SMRT sequencing wherein both DNA ends are capped with hairpin adapters. A SMRTbell template is topologically circular and structurally linear.

SMRT Cell

A flow cell comprising arrays of zero-mode waveguide nanostructures used during Pacific Biosciences SMRT sequencing.

Zero-mode waveguides

Nanophotonic devices that confine light to a small observation volume and are part of the SMRT Cell used during Pacific Biosciences SMRT sequencing.

Flow cell

A disposable component of short-read and long-read sequencing platforms that houses the chemistry to sequence DNA and/or RNA molecules.

Subreads

The sequence derived from a single pass of the DNA polymerase as it processes along the SMRTbell template multiple times during Pacific Biosciences SMRT sequencing. Subreads do not contain any adapter sequences.

Homopolymers

Sequences of consecutive identical bases.

Single-pass

The traversal of a single strand within a SMRTbell template by a DNA polymerase during Pacific Biosciences SMRT sequencing.

Polishing tools

Computational tools that increase genome assembly quality and accuracy. These tools typically compare reads to an assembly to derive a more accurate consensus sequence.

Squiggle

A series of voltage shifts that represent overlapping k-mers from a DNA molecule as it translocates through a nanopore during Oxford Nanopore Technologies sequencing.

Sequencing coverage

The average number of unique reads that align to, or ‘cover’, a sequence or genome.

Circular consensus sequencing

(CCS). A sequencing mode used by Pacific Biosciences in which a DNA polymerase makes multiple passes around the SMRTbell template, generating noisy subreads that are computationally combined to generate a highly accurate high-fidelity consensus read.

Polymerase reads

The sequence derived from one or more passes of the DNA polymerase around a SMRTbell template, including both adapters and inserts. Polymerase reads are trimmed to exclude any low-quality regions and are generated by Pacific Biosciences SMRT sequencing.

Read N50

The sequence length of the shortest read at 50% of the total sequencing dataset sorted by read length. In other words, half of the sequencing dataset is in reads larger than or equal to the read N50 size.

ONT long read

A read that is 10–100 kb in length and generated by Oxford Nanopore Technology (ONT) sequencing.

ONT ultra-long read

A read that is greater than 100 kb in length and generated by Oxford Nanopore Technology (ONT) sequencing.

Contig N50

The sequence length of the shortest contig at 50% of the total genome length sorted by contig length. In other words, half of the genome sequence is contained in contigs larger than or equal to the contig N50 size.

Optical mapping

A technique commonly used to scaffold sequence contigs that involves constructing ordered genomic maps from single molecules of DNA with a fluorescent readout.

Electronic mapping

A technique commonly used to scaffold sequence contigs that involves constructing ordered genomic maps from single molecules of DNA with an electronic readout.

Phased de novo genome assembly

A genome assembly in which the maternal and paternal haplotypes are resolved.

Trio binning

A method in which short reads from two parental genomes are used to partition long reads from their offspring into haplotype-specific sets before the assembly of each haplotype.

Paralogous sequence variants

Single nucleotide differences between duplicated loci in the genome that are invariant in a population.

CHM13 human genome

A complete hydatidiform mole (CHM) genome that has lost the maternal genome and duplicated the paternal genome. This genome is currently the focus of the Telomere-to-Telomere (T2T) consortium's genome assembly efforts due to its essentially haploid nature and stable karyotype.

Whole-genome sequencing

Sequencing of the entire genome without using methods for sequencing selection.

SVA

A type of retrotransposon insertion composed of a (CCCTCT)n hexamer simple repeat region at the 5′ end, an Alu-like region, a variable number of tandem repeat (VNTR) region, a short interspersed element of retroviral origin (SINE-R) region, and a poly(A) tail after the putative polyadenylation signal.

Uniparental disomy

Inheritance of two copies of a chromosome or segments of a chromosome from one parent, instead of one copy from each parent.

Expression quantitative trait loci

Loci that explain a fraction of the genetic variant of a gene expression phenotype.

Genome-wide association studies

An approach used in genetics research to associate specific genetic variations with particular traits.

Introgression

The transfer of genetic information from one species to another as a result of hybridization between them and repeat backcrossing.

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Logsdon, G.A., Vollger, M.R. & Eichler, E.E. Long-read human genome sequencing and its applications. Nat Rev Genet 21, 597–614 (2020). https://doi.org/10.1038/s41576-020-0236-x

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