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VirION2: a short- and long-read sequencing and informatics workflow to study the genomic diversity of viruses in nature
Microbes play fundamental roles in shaping natural ecosystem properties and functions, but do so under constraints imposed by their viral predators. However, studying viruses in nature can be challenging due to low biomass and the lack of universal gene markers. Though metagenomic short-read sequenc...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
PeerJ Inc.
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018248/ https://www.ncbi.nlm.nih.gov/pubmed/33850654 http://dx.doi.org/10.7717/peerj.11088 |
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author | Zablocki, Olivier Michelsen, Michelle Burris, Marie Solonenko, Natalie Warwick-Dugdale, Joanna Ghosh, Romik Pett-Ridge, Jennifer Sullivan, Matthew B. Temperton, Ben |
author_facet | Zablocki, Olivier Michelsen, Michelle Burris, Marie Solonenko, Natalie Warwick-Dugdale, Joanna Ghosh, Romik Pett-Ridge, Jennifer Sullivan, Matthew B. Temperton, Ben |
author_sort | Zablocki, Olivier |
collection | PubMed |
description | Microbes play fundamental roles in shaping natural ecosystem properties and functions, but do so under constraints imposed by their viral predators. However, studying viruses in nature can be challenging due to low biomass and the lack of universal gene markers. Though metagenomic short-read sequencing has greatly improved our virus ecology toolkit—and revealed many critical ecosystem roles for viruses—microdiverse populations and fine-scale genomic traits are missed. Some of these microdiverse populations are abundant and the missed regions may be of interest for identifying selection pressures that underpin evolutionary constraints associated with hosts and environments. Though long-read sequencing promises complete virus genomes on single reads, it currently suffers from high DNA requirements and sequencing errors that limit accurate gene prediction. Here we introduce VirION2, an integrated short- and long-read metagenomic wet-lab and informatics pipeline that updates our previous method (VirION) to further enhance the utility of long-read viral metagenomics. Using a viral mock community, we first optimized laboratory protocols (polymerase choice, DNA shearing size, PCR cycling) to enable 76% longer reads (now median length of 6,965 bp) from 100-fold less input DNA (now 1 nanogram). Using a virome from a natural seawater sample, we compared viromes generated with VirION2 against other library preparation options (unamplified, original VirION, and short-read), and optimized downstream informatics for improved long-read error correction and assembly. VirION2 assemblies combined with short-read based data (‘enhanced’ viromes), provided significant improvements over VirION libraries in the recovery of longer and more complete viral genomes, and our optimized error-correction strategy using long- and short-read data achieved 99.97% accuracy. In the seawater virome, VirION2 assemblies captured 5,161 viral populations (including all of the virus populations observed in the other assemblies), 30% of which were uniquely assembled through inclusion of long-reads, and 22% of the top 10% most abundant virus populations derived from assembly of long-reads. Viral populations unique to VirION2 assemblies had significantly higher microdiversity means, which may explain why short-read virome approaches failed to capture them. These findings suggest the VirION2 sample prep and workflow can help researchers better investigate the virosphere, even from challenging low-biomass samples. Our new protocols are available to the research community on protocols.io as a ‘living document’ to facilitate dissemination of updates to keep pace with the rapid evolution of long-read sequencing technology. |
format | Online Article Text |
id | pubmed-8018248 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | PeerJ Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-80182482021-04-12 VirION2: a short- and long-read sequencing and informatics workflow to study the genomic diversity of viruses in nature Zablocki, Olivier Michelsen, Michelle Burris, Marie Solonenko, Natalie Warwick-Dugdale, Joanna Ghosh, Romik Pett-Ridge, Jennifer Sullivan, Matthew B. Temperton, Ben PeerJ Bioinformatics Microbes play fundamental roles in shaping natural ecosystem properties and functions, but do so under constraints imposed by their viral predators. However, studying viruses in nature can be challenging due to low biomass and the lack of universal gene markers. Though metagenomic short-read sequencing has greatly improved our virus ecology toolkit—and revealed many critical ecosystem roles for viruses—microdiverse populations and fine-scale genomic traits are missed. Some of these microdiverse populations are abundant and the missed regions may be of interest for identifying selection pressures that underpin evolutionary constraints associated with hosts and environments. Though long-read sequencing promises complete virus genomes on single reads, it currently suffers from high DNA requirements and sequencing errors that limit accurate gene prediction. Here we introduce VirION2, an integrated short- and long-read metagenomic wet-lab and informatics pipeline that updates our previous method (VirION) to further enhance the utility of long-read viral metagenomics. Using a viral mock community, we first optimized laboratory protocols (polymerase choice, DNA shearing size, PCR cycling) to enable 76% longer reads (now median length of 6,965 bp) from 100-fold less input DNA (now 1 nanogram). Using a virome from a natural seawater sample, we compared viromes generated with VirION2 against other library preparation options (unamplified, original VirION, and short-read), and optimized downstream informatics for improved long-read error correction and assembly. VirION2 assemblies combined with short-read based data (‘enhanced’ viromes), provided significant improvements over VirION libraries in the recovery of longer and more complete viral genomes, and our optimized error-correction strategy using long- and short-read data achieved 99.97% accuracy. In the seawater virome, VirION2 assemblies captured 5,161 viral populations (including all of the virus populations observed in the other assemblies), 30% of which were uniquely assembled through inclusion of long-reads, and 22% of the top 10% most abundant virus populations derived from assembly of long-reads. Viral populations unique to VirION2 assemblies had significantly higher microdiversity means, which may explain why short-read virome approaches failed to capture them. These findings suggest the VirION2 sample prep and workflow can help researchers better investigate the virosphere, even from challenging low-biomass samples. Our new protocols are available to the research community on protocols.io as a ‘living document’ to facilitate dissemination of updates to keep pace with the rapid evolution of long-read sequencing technology. PeerJ Inc. 2021-03-30 /pmc/articles/PMC8018248/ /pubmed/33850654 http://dx.doi.org/10.7717/peerj.11088 Text en ©2021 Zablocki et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited. |
spellingShingle | Bioinformatics Zablocki, Olivier Michelsen, Michelle Burris, Marie Solonenko, Natalie Warwick-Dugdale, Joanna Ghosh, Romik Pett-Ridge, Jennifer Sullivan, Matthew B. Temperton, Ben VirION2: a short- and long-read sequencing and informatics workflow to study the genomic diversity of viruses in nature |
title | VirION2: a short- and long-read sequencing and informatics workflow to study the genomic diversity of viruses in nature |
title_full | VirION2: a short- and long-read sequencing and informatics workflow to study the genomic diversity of viruses in nature |
title_fullStr | VirION2: a short- and long-read sequencing and informatics workflow to study the genomic diversity of viruses in nature |
title_full_unstemmed | VirION2: a short- and long-read sequencing and informatics workflow to study the genomic diversity of viruses in nature |
title_short | VirION2: a short- and long-read sequencing and informatics workflow to study the genomic diversity of viruses in nature |
title_sort | virion2: a short- and long-read sequencing and informatics workflow to study the genomic diversity of viruses in nature |
topic | Bioinformatics |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018248/ https://www.ncbi.nlm.nih.gov/pubmed/33850654 http://dx.doi.org/10.7717/peerj.11088 |
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