Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.

Natural environments represent an incredible source of microbial genetic diversity. Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of cl...

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Main Authors: Mária Džunková, Giuseppe D'Auria, David Pérez-Villarroya, Andrés Moya
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2012-01-01
Series:PLoS ONE
Online Access:http://europepmc.org/articles/PMC3474745?pdf=render
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author Mária Džunková
Giuseppe D'Auria
David Pérez-Villarroya
Andrés Moya
author_facet Mária Džunková
Giuseppe D'Auria
David Pérez-Villarroya
Andrés Moya
author_sort Mária Džunková
collection DOAJ
description Natural environments represent an incredible source of microbial genetic diversity. Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be "domesticated" for downstream characterization and application, and this makes screening both laborious and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential; however, unfortunately they would remain unexplored. Knowledge of the clone sequences provides important clues about potential biotechnological application of the clones in the library; however, the sequencing of clones one-by-one would be very time-consuming and expensive. In this study, we characterized the first metagenomic clone library from the feces of a healthy human volunteer, using a method based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. Instead of whole individual clone sequencing, we sequenced 358 clones in a pool. The medium-large insert (7-15 kb) cloning strategy allowed us to assemble these clones correctly, and to assign the clone ends to maintain the link between the position of a living clone in the library and the annotated contig from the 454 assembly. Finally, we found several open reading frames (ORFs) with previously described potential medical application. The proposed approach allows planning ad-hoc biochemical assays for the clones of interest, and the appropriate sub-cloning strategy for gene expression in suitable vectors/hosts.
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spelling doaj.art-ad98131b054b4661a9776f396cef5e5c2022-12-21T22:08:54ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-01710e4765410.1371/journal.pone.0047654Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.Mária DžunkováGiuseppe D'AuriaDavid Pérez-VillarroyaAndrés MoyaNatural environments represent an incredible source of microbial genetic diversity. Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be "domesticated" for downstream characterization and application, and this makes screening both laborious and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential; however, unfortunately they would remain unexplored. Knowledge of the clone sequences provides important clues about potential biotechnological application of the clones in the library; however, the sequencing of clones one-by-one would be very time-consuming and expensive. In this study, we characterized the first metagenomic clone library from the feces of a healthy human volunteer, using a method based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. Instead of whole individual clone sequencing, we sequenced 358 clones in a pool. The medium-large insert (7-15 kb) cloning strategy allowed us to assemble these clones correctly, and to assign the clone ends to maintain the link between the position of a living clone in the library and the annotated contig from the 454 assembly. Finally, we found several open reading frames (ORFs) with previously described potential medical application. The proposed approach allows planning ad-hoc biochemical assays for the clones of interest, and the appropriate sub-cloning strategy for gene expression in suitable vectors/hosts.http://europepmc.org/articles/PMC3474745?pdf=render
spellingShingle Mária Džunková
Giuseppe D'Auria
David Pérez-Villarroya
Andrés Moya
Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.
PLoS ONE
title Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.
title_full Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.
title_fullStr Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.
title_full_unstemmed Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.
title_short Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.
title_sort hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications
url http://europepmc.org/articles/PMC3474745?pdf=render
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AT davidperezvillarroya hybridsequencingapproachappliedtohumanfecalmetagenomicclonelibrariesrevealedcloneswithpotentialbiotechnologicalapplications
AT andresmoya hybridsequencingapproachappliedtohumanfecalmetagenomicclonelibrariesrevealedcloneswithpotentialbiotechnologicalapplications