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An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar.

ABSTRACT: How viruses evolve within hosts can dictate infection outcomes; however, reconstructing this process is challenging. We evaluate our multiplexed amplicon approach, PrimalSeq, to demonstrate how virus concentration, sequencing coverage, primer mismatches, and replicates influence the accuracy of measuring intrahost virus diversity. We develop an experimental protocol and computational tool, iVar, for using PrimalSeq to measure virus diversity using Illumina and compare the results to Oxford Nanopore sequencing. We demonstrate the utility of PrimalSeq by measuring Zika and West Nile virus diversity from varied sample types and show that the accumulation of genetic diversity is influenced by experimental and biological systems.

SUBMITTER: Grubaugh ND

PROVIDER: S-EPMC6325816 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Publications

An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar.

Grubaugh Nathan D ND Gangavarapu Karthik K Quick Joshua J Matteson Nathaniel L NL De Jesus Jaqueline Goes JG Main Bradley J BJ Tan Amanda L AL Paul Lauren M LM Brackney Doug E DE Grewal Saran S Gurfield Nikos N Van Rompay Koen K A KKA Isern Sharon S Michael Scott F SF Coffey Lark L LL Loman Nicholas J NJ Andersen Kristian G KG

Genome biology 20190108 1

How viruses evolve within hosts can dictate infection outcomes; however, reconstructing this process is challenging. We evaluate our multiplexed amplicon approach, PrimalSeq, to demonstrate how virus concentration, sequencing coverage, primer mismatches, and replicates influence the accuracy of measuring intrahost virus diversity. We develop an experimental protocol and computational tool, iVar, for using PrimalSeq to measure virus diversity using Illumina and compare the results to Oxford Nanop ...[more]

PMID: 30621750

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Project description:Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes, with 16S rRNA being the gold standard for bacteria. As for fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an in silico primer evaluation, we compared three amplicons (the ITS1-ITS2 region [ITS1-2], 18S ribosomal small subunit RNA, and the D1/D2 domain of the 26S ribosomal large subunit RNA), using biological samples and a mock community of common fungal species. All three targets allowed for accurate identification of the species present. Nevertheless, high heterogeneity in ITS1-2 length was found, and this caused an overestimation of the abundance of species with a shorter ITS, while both 18S and 26S amplicons allowed for more reliable quantification. We demonstrated that ITS1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities, and efforts should be made to promote the use of different targets in sequencing-based microbial ecology studies.IMPORTANCE Amplicon-sequencing approaches for fungi may rely on different targets affecting the diversity and abundance of the fungal species. An increasing number of studies will address fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analyzing a mock community and several biological samples, we demonstrate that using different amplicon targets may change the results of fungal microbiota analysis, and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.

Dataset Information

An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar.

Publications

An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar.

Similar Datasets

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