Accurate Whole Human Genome Sequencing using Reversible Terminator Chemistry

By D. Bentley, Shankar Balasubramanian, H. Swerdlow, G. P. Smith, J. Milton, Clive Brown, K. Hall, D. Evers, Colin L. Barnes, Helen Bignell and 183 more. Nature, 2008

3,840 citations recorded by the source corpus.

DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally used long (400–800 base pair) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intraspecies genetic variation. Here we report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high-quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30× average depth of paired 35-base reads. We characterize four million single-nucleotide polymorphisms and four hundred thousand structural variants, many of which were previously unknown. Our approach is effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications.

Subfields: Genetics, Molecular Biology, Plant Science

View Paper: https://www.semanticscholar.org/paper/6dad16a6941b204c2f5f95d9cda6d0124d5a1a7b

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Citations in the corpus

A window into third-generation sequencing. (2010, methodology): “While this assembly approach is efficient for accurately identifying SNPs in the human genome (44,45), it does not enable a thorough characterization of structural variations, insertions and deletions.”
Next generation sequencing technology: Advances and applications. (2014, background): “1A; step 6) and to block all 3′ ends with ddNTP to prevent the otherwise open 3′ ends to act as sequencing primer sites on adjacent library molecules [4].”
Effective Preparation of Plasmodium vivax Field Isolates for High-Throughput Whole Genome Sequencing (2013, positive): “Certainly highthroughput sequencing technologies [12–14] now provide exciting opportunities to undertake affordable, high-depth whole-genome sequencing of hundreds of Plasmodium parasites.”
Relating the metatranscriptome and metagenome of the human gut (2014, methodology): “Illumina DNA-seq libraries were generated as previously described (36) with the following modifications.”
RNA Sequencing Data: Hitchhiker's Guide to Expression Analysis (2018, positive): “Bridge amplification creates dense “clonal clusters” of each cDNA loaded [8].”
Natural genetic variation caused by small insertions and deletions in the human genome. (2011, background): “We also examined five of the personal human genomes that have been sequenced, including four ‘‘healthy’’ genomes (Levy et al. 2007; Bentley et al. 2008; Wang et al. 2008; Wheeler et al. 2008) and the genome of a patient with acute myelogenous leukemia (Ley et al. 2008).”
Performance comparison of benchtop high-throughput sequencing platforms (2012, methodology): “The MiSeq is based on the existing Solexa sequencing-by-synthesis chemistry [6] but has dramatically reduced run times compared to the Illumina HiSeq (fastest run 4 hours versus 1.”
A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries (2011, background): “The cost of DNA sequencing continues to fall, driven by ongoing innovation in sequencing technology [1-4].”