Strategies for the systematic sequencing of complex genomes

ABSTRACT Recent spectacular advances in the technologies and strategies for DNA sequencing have profoundly accelerated the detailed analysis of genomes from myriad organisms. The past few

years alone have seen the publication of near-complete or draft versions of the genome sequence of several well-studied, multicellular organisms — most notably, the human. As well as

providing data of fundamental biological significance, these landmark accomplishments have yielded important strategic insights that are guiding current and future genome-sequencing

projects. KEY POINTS * The genome sequences of several eukaryotic organisms have been reported in recent years, including a yeast (_Saccharomyces cerevisiae_), a nematode (_Caenorhabditis

elegans_), an insect (_Drosophila melanogaster_), a plant (_Arabidopsis thaliana_) and human (_Homo sapiens_). * These spectacular achievements have been associated with a range of technical

advances in basic sequencing methodology, the automation of many of the key steps in the sequencing pipeline, the adoption of industrial-scale experimental protocols and the development of

improved computational tools for sequence analysis. * The two main strategies used for sequencing large, complex genomes are clone-by-clone shotgun sequencing and whole-genome shotgun

sequencing. Both approaches were used to generate the recently reported working draft human sequences. * In clone-by-clone sequencing, individual clones are selected from a contig map (a

type of physical map) and each is then sequenced by a shotgun-sequencing strategy. In turn, the genome sequence is assembled by pasting together the sequences of the individual clones. * In

whole-genome shotgun sequencing, the genome is broken into fragments of defined size classes, which are then cloned and used to generate sequence reads. In turn, the genome sequence is

assembled from the entire collection of sequence reads. * Each of the two main strategies has strengths and weaknesses, and a hybrid strategy that involves both whole-genome and

clone-by-clone shotgun-sequencing components is being adopted in many current projects. However, it remains to be determined how much sequencing should be done by each strategy when

implementing a hybrid approach. * For new sequencing projects, it is also important to consider whether the genome needs to be sequenced to high accuracy or whether a more draft-level

sequence can provide the information that is required. This consideration will probably influence the choice and implementation of a particular sequencing strategy. * Sequencing the genome

of a complex, multicellular eukaryote still poses massive technological challenges and requires a significant amount of funds. Choosing the appropriate sequencing strategy is therefore a

crucial step in any genome-sequencing project. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access

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subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS BEYOND ASSEMBLY: THE INCREASING FLEXIBILITY OF SINGLE-MOLECULE SEQUENCING TECHNOLOGY Article

09 May 2023 LONG-READ HUMAN GENOME SEQUENCING AND ITS APPLICATIONS Article 05 June 2020 A DRAFT HUMAN PANGENOME REFERENCE Article Open access 10 May 2023 REFERENCES * Green, E. D. in _The

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  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS I thank F. Collins, J. Touchman and R. Wilson for critical reading of this manuscript. AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Genome Technology Branch and NIH Intramural Sequencing Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA Eric D.

Green Authors * Eric D. Green View author publications You can also search for this author inPubMed Google Scholar RELATED LINKS RELATED LINKS FURTHER INFORMATION Human Genome Project

_Saccharomyces cerevisiae_ _Caenorhabditis elegans_ _Drosophila melanogaster_ _Arabidopsis thaliana_ _Homo sapiens_ _Escherichia coli_ Phred Phrap Consed GAP mouse BAC fingerprint map of the

mouse genome rat zebrafish TIGR comprehensive microbial resource Celera Genomics _Tetraodon nigroviridis_ _Fugu rubripes_ rice GLOSSARY * FINISHED SEQUENCE Complete sequence of a clone or

genome, with a defined level of accuracy and contiguity. * SEQUENCE-TAGGED SITE (STS). Short (for example, <1,000 bp), unique sequence associated with a PCR assay that can be used to

detect that site in the genome. * CONTIG Overlapping series of clones or sequence reads (for a clone contig or sequence contig, respectively) that corresponds to a contiguous segment of the

source genome. * MINIMAL TILING PATH A minimal set of overlapping clones that together provides complete coverage across a genomic region. * COVERAGE The average number of times a genomic

segment is represented in a collection of clones or sequence reads (synonymous with redundancy). * SEQUENCE-READY MAP Typically considered an overlapping bacterial clone map (for example, a

BAC contig map) with sufficiently redundant clone coverage to allow for the rational selection of clones for sequencing. * UNIVERSAL PRIMING SITE A short sequence (for example, 16–24 bases)

in a cloning vector, immediately adjacent to the vector–insert junction to which a common (that is, universal) sequencing primer can anneal. * FULL-SHOTGUN SEQUENCE A type of prefinished

sequence, in this case with sufficient coverage to make it ready for sequence finishing (typically on the order of 8–10-fold coverage). * WORKING DRAFT SEQUENCE A type of prefinished

sequence, often meant to correspond to sequence with coverage that puts it at roughly the halfway point towards full-shotgun sequence. * PREFINISHED SEQUENCE Sequence derived from a

preliminary assembly during a shotgun-sequencing project (at this stage, the sequence is often not contiguous nor highly accurate). * RADIATION HYBRID MAP Physical map of markers (typically

STSs) positioned on the basis of the frequency with which they are separated by radiation-induced breaks (map construction involves the PCR analysis of rodent cell lines, each containing

different fragments of the source genome). RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Green, E. Strategies for the systematic sequencing of complex

genomes. _Nat Rev Genet_ 2, 573–583 (2001). https://doi.org/10.1038/35084503 Download citation * Issue Date: 01 August 2001 * DOI: https://doi.org/10.1038/35084503 SHARE THIS ARTICLE Anyone

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