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

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

Rinke, C Low, S Woodcroft, BJ Raina, JB

Skarshewski, A Le, XH Butler, MK Stocker, R Seymour, J

Tyson, GW Hugenholtz, P

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Publication Type:: Journal Article
Citation:: PeerJ, 2016, 2016 (9)
Issue Date:: 2016-01-01

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Field	Value	Language
dc.contributor.author	Rinke, C	en_US
dc.contributor.author	Low, S	en_US
dc.contributor.author	Woodcroft, BJ	en_US
dc.contributor.author	Raina, JB https://orcid.org/0000-0002-7508-0004	en_US
dc.contributor.author	Skarshewski, A	en_US
dc.contributor.author	Le, XH	en_US
dc.contributor.author	Butler, MK	en_US
dc.contributor.author	Stocker, R	en_US
dc.contributor.author	Seymour, J https://orcid.org/0000-0002-3745-6541	en_US
dc.contributor.author	Tyson, GW	en_US
dc.contributor.author	Hugenholtz, P	en_US
dc.date.available	2016-08-24	en_US
dc.date.issued	2016-01-01	en_US
dc.identifier.citation	PeerJ, 2016, 2016 (9)	en_US
dc.identifier.uri	http://hdl.handle.net/10453/53735
dc.description.abstract	© 2016 Rinke et al. 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.	en_US
dc.relation.ispartof	PeerJ	en_US
dc.relation.isbasedon	10.7717/peerj.2486	en_US
dc.title	Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics	en_US
dc.type	Journal Article
utslib.citation.volume	9	en_US
utslib.citation.volume	2016	en_US
utslib.for	0605 Microbiology	en_US
utslib.for	06 Biological Sciences	en_US
utslib.for	11 Medical and Health Sciences	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Strength - C3 - Climate Change Cluster
utslib.copyright.status	open_access
pubs.issue	9	en_US
pubs.publication-status	Published	en_US
pubs.volume	2016	en_US

Abstract:

© 2016 Rinke et al. 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.

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http://hdl.handle.net/10453/53735