Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.

High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metageno...

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Autores principales:	Kathy N Lam, Michael W Hall, Katja Engel, Gregory Vey, Jiujun Cheng, Josh D Neufeld, Trevor C Charles
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/79fb13d6adfa425e8294025766587843
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spelling	oai:doaj.org-article:79fb13d6adfa425e82940257665878432021-11-18T08:16:39ZEvaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.1932-620310.1371/journal.pone.0098968https://doaj.org/article/79fb13d6adfa425e82940257665878432014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24911009/?tool=EBIhttps://doaj.org/toc/1932-6203High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones.Kathy N LamMichael W HallKatja EngelGregory VeyJiujun ChengJosh D NeufeldTrevor C CharlesPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 6, p e98968 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Kathy N Lam Michael W Hall Katja Engel Gregory Vey Jiujun Cheng Josh D Neufeld Trevor C Charles Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.
description	High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones.
format	article
author	Kathy N Lam Michael W Hall Katja Engel Gregory Vey Jiujun Cheng Josh D Neufeld Trevor C Charles
author_facet	Kathy N Lam Michael W Hall Katja Engel Gregory Vey Jiujun Cheng Josh D Neufeld Trevor C Charles
author_sort	Kathy N Lam
title	Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.
title_short	Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.
title_full	Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.
title_fullStr	Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.
title_full_unstemmed	Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.
title_sort	evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/79fb13d6adfa425e8294025766587843
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Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.

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