An analysis of the feasibility of short read sequencing

Whiteford, Nava; Haslam, Niall; Weber, Gerald; Prügel-Bennett, Adam; Essex, Jonathan W.; Roach, Peter L.; Bradley, Mark; Neylon, Cameron

doi:10.1093/nar/gni170

Cited by 103 publications

(77 citation statements)

References 24 publications

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“…This is in agreement with results from previous studies on the effect of read length on determining unique positions in the genome (Whiteford et al 2005;Chaisson et al 2009). A 20-nt tag length, even when adding 6 nt for a pool identifier, is well within the sequence length output of contemporary high-throughput sequencing machines.…”

Section: Whole Genome Profilingsupporting

confidence: 93%

Sequence-based physical mapping of complex genomes by whole genome profiling

Oeveren

Ruiter²,

Jesse³

et al. 2011

Genome Res.

108

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We present whole genome profiling (WGP), a novel next-generation sequencing-based physical mapping technology for construction of bacterial artificial chromosome (BAC) contigs of complex genomes, using Arabidopsis thaliana as an example. WGP leverages short read sequences derived from restriction fragments of two-dimensionally pooled BAC clones to generate sequence tags. These sequence tags are assigned to individual BAC clones, followed by assembly of BAC contigs based on shared regions containing identical sequence tags. Following in silico analysis of WGP sequence tags and simulation of a map of Arabidopsis chromosome 4 and maize, a WGP map of Arabidopsis thaliana ecotype Columbia was constructed de novo using a six-genome equivalent BAC library. Validation of the WGP map using the Columbia reference sequence confirmed that 350 BAC contigs (98%) were assembled correctly, spanning 97% of the 102-Mb calculated genome coverage. We demonstrate that WGP maps can also be generated for more complex plant genomes and will serve as excellent scaffolds to anchor genetic linkage maps and integrate whole genome sequence data.

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Section: Whole Genome Profilingsupporting

confidence: 93%

Sequence-based physical mapping of complex genomes by whole genome profiling

Oeveren

Ruiter²,

Jesse³

et al. 2011

Genome Res.

108

View full text Add to dashboard Cite

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“…Thus, the percentage coverage does not increase drastically with data from additional flow cells. This is not surprising since only 80% of a complex eukaryotic genome can be uniquely mapped with short 36-bp reads (Whiteford et al 2005). Using this threshold, we identified 165 candidate SNPs that distinguished the mutagenized third chromosome from the unmutagenized chromosome.…”

Section: Resultsmentioning

confidence: 99%

Identification of EMS-Induced Mutations inDrosophila melanogasterby Whole-Genome Sequencing

et al. 2009

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Next-generation methods for rapid whole-genome sequencing enable the identification of single-basepair mutations in Drosophila by comparing a chromosome bearing a new mutation to the unmutagenized sequence. To validate this approach, we sought to identify the molecular lesion responsible for a recessive EMS-induced mutation affecting egg shell morphology by using Illumina next-generation sequencing. After obtaining sufficient sequence from larvae that were homozygous for either wild-type or mutant chromosomes, we obtained high-quality reads for base pairs composing $70% of the third chromosome of both DNA samples. We verified 103 single-base-pair changes between the two chromosomes. Nine changes were nonsynonymous mutations and two were nonsense mutations. One nonsense mutation was in a gene, encore, whose mutations produce an egg shell phenotype also observed in progeny of homozygous mutant mothers. Complementation analysis revealed that the chromosome carried a new functional allele of encore, demonstrating that one round of next-generation sequencing can identify the causative lesion for a phenotype of interest. This new method of whole-genome sequencing represents great promise for mutant mapping in flies, potentially replacing conventional methods. S TANDARD practices of genetic mapping typically occur in three phases. First, polymorphisms that distinguish the chromosome carrying the mutation to be mapped from that of the homolog bearing a wild-type allele of that gene must be identified. Second, by genotyping recombinant chromosomes that do or do not carry the mutation of interest, an association between polymorphisms and the mutation can be identified, which can then be used to pinpoint the location of the relevant mutation. Finally, candidate genes within the interval must be identified and regions sequenced to find the causative mutation. Often, these three steps are performed iteratively. In situations where there are few polymorphic markers or candidate genes, this process can be arduous and, depending on the organism, can consume months to years.New genome-sequencing technologies (Margulies et al. 2005;Bentley 2006;Barski et al. 2007;Sarin et al. 2008;Smith et al. 2008;Valouev et al. 2008) show tremendous promise for reducing the time needed to identify causative mutations. Using these approaches, one may be able to directly identify causative lesions by comparing the nucleotide sequences of wild-type and mutant genomes. Indeed, we have conducted a proofof-principle experiment to determine the feasibility of such an approach in Drosophila melanogaster. In the course of conducting an EMS-based genetic screen, we identified a chromosome, designated 791, which displayed a fused dorsal appendage phenotype in embryos of homozygous mothers. Such phenotypes usually arise from a defect in the maternal establishment of the dorso-ventral axis. To identify the mutated gene that gives rise to this phenotype, we used a next-generation sequencing platform to directly compare the nucleotide sequence of the...

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“…Similarly, the number of uniquely aligned reads is reduced when aligning to larger, more complex genomes or reference sequences because of their having a higher probability of repetitive sequences. A case in point is a modeling study that indicated that 97% of the E. coli genome can be uniquely aligned with 18-bp reads but that only 90% of the human genome can be uniquely aligned with 30-bp reads (68,69 ). Unique alignment or assembly is reduced not only by the presence of repeat sequences but also by shared homologies within closely related gene families and pseudogenes.…”

Section: Ngs Data Analysismentioning

confidence: 99%

Next-Generation Sequencing: From Basic Research to Diagnostics

2009

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Background: For the past 30 years, the Sanger method has been the dominant approach and gold standard for DNA sequencing. The commercial launch of the first massively parallel pyrosequencing platform in 2005 ushered in the new era of high-throughput genomic analysis now referred to as next-generation sequencing (NGS). Content: This review describes fundamental principles of commercially available NGS platforms. Although the platforms differ in their engineering configurations and sequencing chemistries, they share a technical paradigm in that sequencing of spatially separated, clonally amplified DNA templates or single DNA molecules is performed in a flow cell in a massively parallel manner. Through iterative cycles of polymerase-mediated nucleotide extensions or, in one approach, through successive oligonucleotide ligations, sequence outputs in the range of hundreds of megabases to gigabases are now obtained routinely. Highlighted in this review are the impact of NGS on basic research, bioinformatics considerations, and translation of this technology into clinical diagnostics. Also presented is a view into future technologies, including real-time single-molecule DNA sequencing and nanopore-based sequencing. Summary: In the relatively short time frame since 2005, NGS has fundamentally altered genomics research and allowed investigators to conduct experiments that were previously not technically feasible or affordable. The various technologies that constitute this new paradigm continue to evolve, and further improvements in technology robustness and process streamlining will pave the path for translation into clinical diagnostics.

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An analysis of the feasibility of short read sequencing

Cited by 103 publications

References 24 publications

Sequence-based physical mapping of complex genomes by whole genome profiling

Sequence-based physical mapping of complex genomes by whole genome profiling

Identification of EMS-Induced Mutations inDrosophila melanogasterby Whole-Genome Sequencing

Next-Generation Sequencing: From Basic Research to Diagnostics

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