Phylogenetic screening of a bacterial, metagenomic library using homing endonuclease restriction and marker insertion
Identifieur interne : 000104 ( Ncbi/Merge ); précédent : 000103; suivant : 000105Phylogenetic screening of a bacterial, metagenomic library using homing endonuclease restriction and marker insertion
Auteurs : Pui Yi Yung [Australie] ; Catherine Burke [Australie] ; Matt Lewis [États-Unis] ; Suhelen Egan [Australie] ; Staffan Kjelleberg [Australie] ; Torsten Thomas [Australie]Source :
- Nucleic Acids Research [ 0305-1048 ] ; 2009.
Abstract
Metagenomics provides access to the uncultured majority of the microbial world. The approaches employed in this field have, however, had limited success in linking functional genes to the taxonomic or phylogenetic origin of the organism they belong to. Here we present an efficient strategy to recover environmental DNA fragments that contain phylogenetic marker genes from metagenomic libraries. Our method involves the cleavage of 23S ribsosmal RNA (rRNA) genes within pooled library clones by the homing endonuclease I-
Url:
DOI: 10.1093/nar/gkp746
PubMed: 19767618
PubMed Central: 2790883
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PMC:2790883Le document en format XML
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<series><title level="j">Nucleic Acids Research</title>
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<front><div type="abstract" xml:lang="en"><p>Metagenomics provides access to the uncultured majority of the microbial world. The approaches employed in this field have, however, had limited success in linking functional genes to the taxonomic or phylogenetic origin of the organism they belong to. Here we present an efficient strategy to recover environmental DNA fragments that contain phylogenetic marker genes from metagenomic libraries. Our method involves the cleavage of 23S ribsosmal RNA (rRNA) genes within pooled library clones by the homing endonuclease I-<italic>Ceu</italic>
I followed by the insertion and selection of an antibiotic resistance cassette. This approach was applied to screen a library of 6500 fosmid clones derived from the microbial community associated with the sponge <italic>Cymbastela concentrica</italic>
. Several fosmid clones were recovered after the screen and detailed phylogenetic and taxonomic assignment based on the rRNA gene showed that they belong to previously unknown organisms. In addition, compositional features of these fosmid clones were used to classify and taxonomically assign a dataset of environmental shotgun sequences. Our approach represents a valuable tool for the analysis of rapidly increasing, environmental DNA sequencing information.</p>
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<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Nucleic Acids Res</journal-id>
<journal-id journal-id-type="iso-abbrev">Nucleic Acids Res</journal-id>
<journal-id journal-id-type="publisher-id">nar</journal-id>
<journal-id journal-id-type="hwp">nar</journal-id>
<journal-title-group><journal-title>Nucleic Acids Research</journal-title>
</journal-title-group>
<issn pub-type="ppub">0305-1048</issn>
<issn pub-type="epub">1362-4962</issn>
<publisher><publisher-name>Oxford University Press</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">19767618</article-id>
<article-id pub-id-type="pmc">2790883</article-id>
<article-id pub-id-type="doi">10.1093/nar/gkp746</article-id>
<article-id pub-id-type="publisher-id">gkp746</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Methods Online</subject>
</subj-group>
</article-categories>
<title-group><article-title>Phylogenetic screening of a bacterial, metagenomic library using homing endonuclease restriction and marker insertion</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Yung</surname>
<given-names>Pui Yi</given-names>
</name>
<xref ref-type="aff" rid="AFF1"><sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Burke</surname>
<given-names>Catherine</given-names>
</name>
<xref ref-type="aff" rid="AFF1"><sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Lewis</surname>
<given-names>Matt</given-names>
</name>
<xref ref-type="aff" rid="AFF1"><sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Egan</surname>
<given-names>Suhelen</given-names>
</name>
<xref ref-type="aff" rid="AFF1"><sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Kjelleberg</surname>
<given-names>Staffan</given-names>
</name>
<xref ref-type="aff" rid="AFF1"><sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Thomas</surname>
<given-names>Torsten</given-names>
</name>
<xref ref-type="aff" rid="AFF1"><sup>1</sup>
</xref>
<xref ref-type="corresp" rid="COR1">*</xref>
</contrib>
</contrib-group>
<aff id="AFF1"><sup>1</sup>
Centre for Marine Bio-Innovation and School of Biotechnology and Biomolecular Sciences, University of New South Wales, 2035 NSW, Australia and<sup>2</sup>
J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA</aff>
<author-notes><corresp id="COR1">*To whom correspondence should be addressed. Tel: <phone>+61 2 93853467</phone>
; Fax: <fax>+61 2 93851779</fax>
; Email: <email>t.thomas@unsw.edu.au</email>
</corresp>
</author-notes>
<pub-date pub-type="ppub"><month>11</month>
<year>2009</year>
</pub-date>
<pub-date pub-type="epub"><day>18</day>
<month>9</month>
<year>2009</year>
</pub-date>
<pub-date pub-type="pmc-release"><day>18</day>
<month>9</month>
<year>2009</year>
</pub-date>
<pmc-comment> PMC Release delay is 0 months and 0 days and was based on the
. </pmc-comment>
<volume>37</volume>
<issue>21</issue>
<fpage>e144</fpage>
<lpage>e144</lpage>
<history><date date-type="received"><day>1</day>
<month>6</month>
<year>2009</year>
</date>
<date date-type="rev-recd"><day>8</day>
<month>7</month>
<year>2009</year>
</date>
<date date-type="accepted"><day>24</day>
<month>8</month>
<year>2009</year>
</date>
</history>
<permissions><copyright-statement>© The Author(s) 2009. Published by Oxford University Press.</copyright-statement>
<copyright-year>2009</copyright-year>
<license license-type="creative-commons" xlink:href="http://creativecommons.org/licenses/by-nc/2.5/uk/"><license-p><pmc-comment>CREATIVE COMMONS</pmc-comment>
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc/2.5/uk/">http://creativecommons.org/licenses/by-nc/2.5/uk/</ext-link>
) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p>
</license>
</permissions>
<abstract><p>Metagenomics provides access to the uncultured majority of the microbial world. The approaches employed in this field have, however, had limited success in linking functional genes to the taxonomic or phylogenetic origin of the organism they belong to. Here we present an efficient strategy to recover environmental DNA fragments that contain phylogenetic marker genes from metagenomic libraries. Our method involves the cleavage of 23S ribsosmal RNA (rRNA) genes within pooled library clones by the homing endonuclease I-<italic>Ceu</italic>
I followed by the insertion and selection of an antibiotic resistance cassette. This approach was applied to screen a library of 6500 fosmid clones derived from the microbial community associated with the sponge <italic>Cymbastela concentrica</italic>
. Several fosmid clones were recovered after the screen and detailed phylogenetic and taxonomic assignment based on the rRNA gene showed that they belong to previously unknown organisms. In addition, compositional features of these fosmid clones were used to classify and taxonomically assign a dataset of environmental shotgun sequences. Our approach represents a valuable tool for the analysis of rapidly increasing, environmental DNA sequencing information.</p>
</abstract>
</article-meta>
</front>
</pmc>
<affiliations><list><country><li>Australie</li>
<li>États-Unis</li>
</country>
<region><li>Maryland</li>
</region>
</list>
<tree><country name="Australie"><noRegion><name sortKey="Yung, Pui Yi" sort="Yung, Pui Yi" uniqKey="Yung P" first="Pui Yi" last="Yung">Pui Yi Yung</name>
</noRegion>
<name sortKey="Burke, Catherine" sort="Burke, Catherine" uniqKey="Burke C" first="Catherine" last="Burke">Catherine Burke</name>
<name sortKey="Egan, Suhelen" sort="Egan, Suhelen" uniqKey="Egan S" first="Suhelen" last="Egan">Suhelen Egan</name>
<name sortKey="Kjelleberg, Staffan" sort="Kjelleberg, Staffan" uniqKey="Kjelleberg S" first="Staffan" last="Kjelleberg">Staffan Kjelleberg</name>
<name sortKey="Thomas, Torsten" sort="Thomas, Torsten" uniqKey="Thomas T" first="Torsten" last="Thomas">Torsten Thomas</name>
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<country name="États-Unis"><region name="Maryland"><name sortKey="Lewis, Matt" sort="Lewis, Matt" uniqKey="Lewis M" first="Matt" last="Lewis">Matt Lewis</name>
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