Dataset Information

Human Genome Sequencing at the Population Scale: A Primer on High-Throughput DNA Sequencing and Analysis.

ABSTRACT: Most human diseases have underlying genetic causes. To better understand the impact of genes on disease and its implications for medicine and public health, researchers have pursued methods for determining the sequences of individual genes, then all genes, and now complete human genomes. Massively parallel high-throughput sequencing technology, where DNA is sheared into smaller pieces, sequenced, and then computationally reordered and analyzed, enables fast and affordable sequencing of full human genomes. As the price of sequencing continues to decline, more and more individuals are having their genomes sequenced. This may facilitate better population-level disease subtyping and characterization, as well as individual-level diagnosis and personalized treatment and prevention plans. In this review, we describe several massively parallel high-throughput DNA sequencing technologies and their associated strengths, limitations, and error modes, with a focus on applications in epidemiologic research and precision medicine. We detail the methods used to computationally process and interpret sequence data to inform medical or preventative action.

SUBMITTER: Goldfeder RL

PROVIDER: S-EPMC6250075 | biostudies-other | 2017 Oct

REPOSITORIES: biostudies-other

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Human Genome Sequencing at the Population Scale: A Primer on High-Throughput DNA Sequencing and Analysis.

Goldfeder Rachel L RL Wall Dennis P DP Khoury Muin J MJ Ioannidis John P A JPA Ashley Euan A EA

American journal of epidemiology 20171001 8

Most human diseases have underlying genetic causes. To better understand the impact of genes on disease and its implications for medicine and public health, researchers have pursued methods for determining the sequences of individual genes, then all genes, and now complete human genomes. Massively parallel high-throughput sequencing technology, where DNA is sheared into smaller pieces, sequenced, and then computationally reordered and analyzed, enables fast and affordable sequencing of full huma ...[more]

PMID: 29040395

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Project description:BackgroundIn a high-throughput environment, to PCR amplify and sequence a large set of viral isolates from populations that are potentially heterogeneous and continuously evolving, the use of degenerate PCR primers is an important strategy. Degenerate primers allow for the PCR amplification of a wider range of viral isolates with only one set of pre-mixed primers, thus increasing amplification success rates and minimizing the necessity for genome finishing activities. To successfully select a large set of degenerate PCR primers necessary to tile across an entire viral genome and maximize their success, this process is best performed computationally.ResultsWe have developed a fully automated degenerate PCR primer design system that plays a key role in the J. Craig Venter Institute's (JCVI) high-throughput viral sequencing pipeline. A consensus viral genome, or a set of consensus segment sequences in the case of a segmented virus, is specified using IUPAC ambiguity codes in the consensus template sequence to represent the allelic diversity of the target population. PCR primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the full length of the specified target region. As part of the tiling process, primer pairs are computationally screened to meet the criteria for successful PCR with one of two described amplification protocols. The actual sequencing success rates for designed primers for measles virus, mumps virus, human parainfluenza virus 1 and 3, human respiratory syncytial virus A and B and human metapneumovirus are described, where >90% of designed primer pairs were able to consistently successfully amplify >75% of the isolates.ConclusionsAugmenting our previously developed and published JCVI Primer Design Pipeline, we achieved similarly high sequencing success rates with only minor software modifications. The recommended methodology for the construction of the consensus sequence that encapsulates the allelic variation of the targeted population and is a key step prior to designing degenerate primers is also formally described.

Dataset Information

Human Genome Sequencing at the Population Scale: A Primer on High-Throughput DNA Sequencing and Analysis.

Publications

Human Genome Sequencing at the Population Scale: A Primer on High-Throughput DNA Sequencing and Analysis.

Similar Datasets

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