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

Rinke, Christian, Low, Serene, Woodcroft, Ben J., Raina, Jean-Baptista, Skarshewski, Adam, Le, Xuyen H., Butler, Margaret K., Stocker, Roman, Seymour, Justin, Tyson, Gene W. and Hugenholtz, Philip (2016) Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics. PeerJ, 4 1-28. doi:10.7717/peerj.2486


Author Rinke, Christian
Low, Serene
Woodcroft, Ben J.
Raina, Jean-Baptista
Skarshewski, Adam
Le, Xuyen H.
Butler, Margaret K.
Stocker, Roman
Seymour, Justin
Tyson, Gene W.
Hugenholtz, Philip
Title Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics
Journal name PeerJ   Check publisher's open access policy
ISSN 2167-8359
Publication date 2016-09-22
Year available 2016
Sub-type Article (original research)
DOI 10.7717/peerj.2486
Open Access Status DOI
Volume 4
Start page 1
End page 28
Total pages 28
Place of publication London, United Kingdom
Publisher PeerJ
Language eng
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.
Keyword Nextera XT
100 fg
Low input DNA library
Picogram
Reagent contamination
Low biomass
Low volume
Microscale metagenomics
Marine microheterogeneity
Illumina
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID GBMF3801
FL150100038
DE-SC0004632
DE160100248
Institutional Status UQ

 
Versions
Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 4 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 6 times in Scopus Article | Citations
Google Scholar Search Google Scholar
Created: Fri, 07 Oct 2016, 21:03:29 EST by Mrs Louise Nimwegen on behalf of School of Chemistry & Molecular Biosciences