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Rinke C, Low S, Woodcroft BJ, et al. Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics. PeerJ. 2016;4:e2486doi: 10.7717/peerj.2486.
Rinke, C., Low, S., Woodcroft, B. J., Raina, J. B., Skarshewski, A., Le, X. H., Butler, M. K., Stocker, R., Seymour, J., Tyson, G. W., & Hugenholtz, P. (2016). Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics. PeerJ, 4e2486. https://doi.org/10.7717/peerj.2486
Rinke, Christian, et al. "Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics." PeerJ vol. 4 (2016): e2486. doi: https://doi.org/10.7717/peerj.2486
Rinke C, Low S, Woodcroft BJ, Raina JB, Skarshewski A, Le XH, Butler MK, Stocker R, Seymour J, Tyson GW, Hugenholtz P. Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics. PeerJ. 2016 Sep 22;4:e2486. doi: 10.7717/peerj.2486. eCollection 2016. PMID: 27688978; PMCID: PMC5036114.
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PeerJ. 2016 Sep 22;4:e2486. doi: 10.7717/peerj.2486. eCollection 2016.

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

PeerJ

Christian Rinke, Serene Low, Ben J Woodcroft, Jean-Baptiste Raina, Adam Skarshewski, Xuyen H Le, Margaret K Butler, Roman Stocker, Justin Seymour, Gene W Tyson, Philip Hugenholtz

Affiliations

  1. Australian Centre for Ecogenomics/School of Chemistry and Molecular Biosciences, University of Queensland , Brisbane, QLD , Australia.
  2. Climate Change Cluster, University of Technology Sydney , Sydney, New South Wales , Australia.
  3. Department of Civil, Environmental and Geomatic Engineering, ETH Zurich , Zurich , Switzerland.
  4. Australian Centre for Ecogenomics/School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, Australia; Advanced Water Management Centre, University of Queensland, Brisbane, QLD, Australia.
  5. Australian Centre for Ecogenomics/School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, Australia; Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia.

PMID: 27688978 PMCID: PMC5036114 DOI: 10.7717/peerj.2486

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. Here 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.

Keywords: 100 fg; Illumina; Low biomass; Low input DNA library; Low volume; Marine microheterogeneity; Microscale metagenomics; Nextera XT; Picogram; Reagent contamination

Conflict of interest statement

The authors declare that they have no competing interests.

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