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...
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| Format: | Article |
| Language: | English |
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PeerJ Inc.
2016-09-01
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| Online Access: | https://peerj.com/articles/2486.pdf |
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| author | 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 |
| author_facet | 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 |
| author_sort | Christian Rinke |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-09T06:21:46Z |
| format | Article |
| id | doaj.art-454b85ebe8b84875a55ee53148eeb875 |
| institution | Directory Open Access Journal |
| issn | 2167-8359 |
| language | English |
| last_indexed | 2024-03-09T06:21:46Z |
| publishDate | 2016-09-01 |
| publisher | PeerJ Inc. |
| record_format | Article |
| series | PeerJ |
| spelling | doaj.art-454b85ebe8b84875a55ee53148eeb8752023-12-03T11:34:37ZengPeerJ Inc.PeerJ2167-83592016-09-014e248610.7717/peerj.2486Validation of picogram- and femtogram-input DNA libraries for microscale metagenomicsChristian Rinke0Serene Low1Ben J. Woodcroft2Jean-Baptiste Raina3Adam Skarshewski4Xuyen H. Le5Margaret K. Butler6Roman Stocker7Justin Seymour8Gene W. Tyson9Philip Hugenholtz10Australian Centre for Ecogenomics/School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, AustraliaAustralian Centre for Ecogenomics/School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, AustraliaAustralian Centre for Ecogenomics/School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, AustraliaClimate Change Cluster, University of Technology Sydney, Sydney, New South Wales, AustraliaAustralian Centre for Ecogenomics/School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, AustraliaAustralian Centre for Ecogenomics/School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, AustraliaAustralian Centre for Ecogenomics/School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, AustraliaDepartment of Civil, Environmental and Geomatic Engineering, ETH Zurich, Zurich, SwitzerlandClimate Change Cluster, University of Technology Sydney, Sydney, New South Wales, AustraliaAustralian Centre for Ecogenomics/School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, AustraliaAustralian Centre for Ecogenomics/School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, AustraliaHigh-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.https://peerj.com/articles/2486.pdfNextera XT100 fgLow input DNA libraryPicogramReagent contaminationLow biomass |
| spellingShingle | 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 Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics PeerJ Nextera XT 100 fg Low input DNA library Picogram Reagent contamination Low biomass |
| title | Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics |
| title_full | Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics |
| title_fullStr | Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics |
| title_full_unstemmed | Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics |
| title_short | Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics |
| title_sort | validation of picogram and femtogram input dna libraries for microscale metagenomics |
| topic | Nextera XT 100 fg Low input DNA library Picogram Reagent contamination Low biomass |
| url | https://peerj.com/articles/2486.pdf |
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