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

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... Full description

Journal Title: PeerJ 01 September 2016, Vol.4, p.e2486
Main Author: Christian Rinke
Other Authors: 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
Format: Electronic Article Electronic Article
Language: English
Subjects:
ID: E-ISSN: 2167-8359 ; DOI: 10.7717/peerj.2486
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title: Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics
format: Article
creator:
  • 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
subjects:
  • Nextera Xt
  • 100 Fg
  • Low Input DNA Library
  • Picogram
  • Reagent Contamination
  • Low Biomass
  • Medicine
ispartof: PeerJ, 01 September 2016, Vol.4, p.e2486
description: 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.
language: eng
source:
identifier: E-ISSN: 2167-8359 ; DOI: 10.7717/peerj.2486
fulltext: fulltext_linktorsrc
issn:
  • 2167-8359
  • 21678359
url: Link


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titleValidation of picogram- and femtogram-input DNA libraries for microscale metagenomics
creatorChristian 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
ispartofPeerJ, 01 September 2016, Vol.4, p.e2486
identifierE-ISSN: 2167-8359 ; DOI: 10.7717/peerj.2486
subjectNextera Xt ; 100 Fg ; Low Input DNA Library ; Picogram ; Reagent Contamination ; Low Biomass ; Medicine
descriptionHigh-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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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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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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