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Title: Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics

Abstract

High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. For this study, we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diverse Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (~100–1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities.more » We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics.« less

Authors:
 [1];  [1]; ORCiD logo [1];  [2];  [1];  [1];  [1];  [3];  [2];  [4];  [5]
  1. Univ. of Queensland, Brisbane (Australia). Australian Centre for Ecogenomics and School of Chemistry and Molecular Biosciences
  2. Univ. of Technology Sydney, NSW (Australia). Climate Change Cluster
  3. ETH Zurich (Switzerland). Dept. of Civil, Environmental, and Geomatic Engineering
  4. Univ. of Queensland, Brisbane (Australia). Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences, and Advanced Water Management Centre
  5. Univ. of Queensland, Brisbane (Australia). Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences and Inst. for Molecular Bioscience
Publication Date:
Research Org.:
Univ. of Arizona, Tucson, AZ (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); Gordon and Betty Moore Foundation; Australian Research Council (ARC)
OSTI Identifier:
1435770
Grant/Contract Number:  
SC0004632; GBMF3801; FL150100038; 160100248
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
PeerJ
Additional Journal Information:
Journal Volume: 4; Journal ID: ISSN 2167-8359
Publisher:
PeerJ Inc.
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Nextera XT; 100 fg; Low input DNA library; Picogram; Reagent contamination; Low biomass; Low volume; Microscale metagenomics; Marine microheterogeneity; Illumina

Citation Formats

Rinke, Christian, Low, Serene, Woodcroft, Ben J., Raina, Jean-Baptiste, Skarshewski, Adam, Le, Xuyen H., Butler, Margaret K., Stocker, Roman, Seymour, Justin, Tyson, Gene W., and Hugenholtz, Philip. Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics. United States: N. p., 2016. Web. doi:10.7717/peerj.2486.
Rinke, Christian, Low, Serene, Woodcroft, Ben J., Raina, Jean-Baptiste, Skarshewski, Adam, Le, Xuyen H., Butler, Margaret K., Stocker, Roman, Seymour, Justin, Tyson, Gene W., & Hugenholtz, Philip. Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics. United States. doi:10.7717/peerj.2486.
Rinke, Christian, Low, Serene, Woodcroft, Ben J., Raina, Jean-Baptiste, Skarshewski, Adam, Le, Xuyen H., Butler, Margaret K., Stocker, Roman, Seymour, Justin, Tyson, Gene W., and Hugenholtz, Philip. Thu . "Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics". United States. doi:10.7717/peerj.2486. https://www.osti.gov/servlets/purl/1435770.
@article{osti_1435770,
title = {Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics},
author = {Rinke, Christian and Low, Serene and Woodcroft, Ben J. and Raina, Jean-Baptiste and Skarshewski, Adam and Le, Xuyen H. and Butler, Margaret K. and Stocker, Roman and Seymour, Justin and Tyson, Gene W. and Hugenholtz, Philip},
abstractNote = {High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. For this study, we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diverse Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (~100–1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics.},
doi = {10.7717/peerj.2486},
journal = {PeerJ},
issn = {2167-8359},
number = ,
volume = 4,
place = {United States},
year = {2016},
month = {9}
}

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Cited by: 11 works
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Works referenced in this record:

WebLogo: A Sequence Logo Generator
journal, May 2004

  • Crooks, Gavin E.; Hon, Gary; Chandonia, John-Marc
  • Genome Research, Vol. 14, Issue 6, p. 1188-1190
  • DOI: 10.1101/gr.849004