Dataset Information

A Systematic Evaluation of High-Throughput Sequencing Approaches to Identify Low-Frequency Single Nucleotide Variants in Viral Populations.

ABSTRACT: High-throughput sequencing such as those provided by Illumina are an efficient way to understand sequence variation within viral populations. However, challenges exist in distinguishing process-introduced error from biological variance, which significantly impacts our ability to identify sub-consensus single-nucleotide variants (SNVs). Here we have taken a systematic approach to evaluate laboratory and bioinformatic pipelines to accurately identify low-frequency SNVs in viral populations. Artificial DNA and RNA "populations" were created by introducing known SNVs at predetermined frequencies into template nucleic acid before being sequenced on an Illumina MiSeq platform. These were used to assess the effects of abundance and starting input material type, technical replicates, read length and quality, short-read aligner, and percentage frequency thresholds on the ability to accurately call variants. Analyses revealed that the abundance and type of input nucleic acid had the greatest impact on the accuracy of SNV calling as measured by a micro-averaged Matthews correlation coefficient score, with DNA and high RNA inputs (107 copies) allowing for variants to be called at a 0.2% frequency. Reduced input RNA (105 copies) required more technical replicates to maintain accuracy, while low RNA inputs (103 copies) suffered from consensus-level errors. Base errors identified at specific motifs identified in all technical replicates were also identified which can be excluded to further increase SNV calling accuracy. These findings indicate that samples with low RNA inputs should be excluded for SNV calling and reinforce the importance of optimising the technical and bioinformatics steps in pipelines that are used to accurately identify sequence variants.

SUBMITTER: King DJ

PROVIDER: S-EPMC7594041 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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A Systematic Evaluation of High-Throughput Sequencing Approaches to Identify Low-Frequency Single Nucleotide Variants in Viral Populations.

King David J DJ Freimanis Graham G Lasecka-Dykes Lidia L Asfor Amin A Ribeca Paolo P Waters Ryan R King Donald P DP Laing Emma E

Viruses 20201020 10

High-throughput sequencing such as those provided by Illumina are an efficient way to understand sequence variation within viral populations. However, challenges exist in distinguishing process-introduced error from biological variance, which significantly impacts our ability to identify sub-consensus single-nucleotide variants (SNVs). Here we have taken a systematic approach to evaluate laboratory and bioinformatic pipelines to accurately identify low-frequency SNVs in viral populations. Artifi ...[more]

PMID: 33092085

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Project description:BACKGROUND:Sensitive detection of low-frequency single nucleotide variants carries great significance in many applications. In cancer genetics research, tumor biopsies are a mixture of normal and tumor cells from various subpopulations due to tumor heterogeneity. Thus the frequencies of somatic variants from a subpopulation tend to be low. Liquid biopsies, which monitor circulating tumor DNA in blood to detect metastatic potential, also face the challenge of detecting low-frequency variants due to the small percentage of the circulating tumor DNA in blood. Moreover, in population genetics research, although pooled sequencing of a large number of individuals is cost-effective, pooling dilutes the signals of variants from any individual. Detection of low frequency variants is difficult and can be cofounded by sequencing artifacts. Existing methods are limited in sensitivity and mainly focus on frequencies around 2 % to 5 %; most fail to consider differential sequencing artifacts. RESULTS:We aimed to push down the frequency detection limit close to the position specific sequencing error rates by modeling the observed erroneous read counts with respect to genomic sequence contexts. 4 distributions suitable for count data modeling (using generalized linear models) were extensively characterized in terms of their goodness-of-fit as well as the performances on real sequencing data benchmarks, which were specifically designed for testing detection of low-frequency variants; two sequencing technologies with significantly different chemistry mechanisms were used to explore systematic errors. We found the zero-inflated negative binomial distribution generalized linear mode is superior to the other models tested, and the advantage is most evident at 0.5 % to 1 % range. This method is also generalizable to different sequencing technologies. Under standard sequencing protocols and depth given in the testing benchmarks, 95.3 % recall and 79.9 % precision for Ion Proton data, 95.6 % recall and 97.0 % precision for Illumina MiSeq data were achieved for SNVs with frequency?>?= 1 %, while the detection limit is around 0.5 %. CONCLUSIONS:Our method enables sensitive detection of low-frequency single nucleotide variants across different sequencing platforms and will facilitate research and clinical applications such as pooled sequencing, cancer early detection, prognostic assessment, metastatic monitoring, and relapses or acquired resistance identification.

Dataset Information

A Systematic Evaluation of High-Throughput Sequencing Approaches to Identify Low-Frequency Single Nucleotide Variants in Viral Populations.

Publications

A Systematic Evaluation of High-Throughput Sequencing Approaches to Identify Low-Frequency Single Nucleotide Variants in Viral Populations.

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