Direct 16S rRNA-seq from bacterial communities: a PCR-independent approach to simultaneously assess microbial diversity and functional activity potential of each taxon
Identifieur interne : 000146 ( Pmc/Curation ); précédent : 000145; suivant : 000147Direct 16S rRNA-seq from bacterial communities: a PCR-independent approach to simultaneously assess microbial diversity and functional activity potential of each taxon
Auteurs : Riccardo Rosselli [Italie] ; Ottavia Romoli [Italie] ; Nicola Vitulo [Italie] ; Alessandro Vezzi [Italie] ; Stefano Campanaro [Italie] ; Fabio De Pascale [Italie] ; Riccardo Schiavon [Italie] ; Maurizio Tiarca [Italie] ; Fabio Poletto [Italie] ; Giuseppe Concheri [Italie] ; Giorgio Valle [Italie] ; Andrea Squartini [Italie]Source :
- Scientific Reports [ 2045-2322 ] ; 2016.
Abstract
The analysis of environmental microbial communities has largely relied on a PCR-dependent amplification of genes entailing species identity as 16S rRNA. This approach is susceptible to biases depending on the level of primer matching in different species. Moreover, possible yet-to-discover taxa whose rRNA could differ enough from known ones would not be revealed. DNA-based methods moreover do not provide information on the actual physiological relevance of each taxon within an environment and are affected by the variable number of rRNA operons in different genomes. To overcome these drawbacks we propose an approach of direct sequencing of 16S ribosomal RNA without any primer- or PCR-dependent step. The method was tested on a microbial community developing in an anammox bioreactor sampled at different time-points. A conventional PCR-based amplicon pyrosequencing was run in parallel. The community resulting from direct rRNA sequencing was highly consistent with the known biochemical processes operative in the reactor. As direct rRNA-seq is based not only on taxon abundance but also on physiological activity, no comparison between its results and those from PCR-based approaches can be applied. The novel principle is in this respect proposed not as an alternative but rather as a complementary methodology in microbial community studies.
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DOI: 10.1038/srep32165
PubMed: 27577787
PubMed Central: 5006002
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strict</wicri:regionArea></affiliation></author><author><name sortKey="Campanaro, Stefano" sort="Campanaro, Stefano" uniqKey="Campanaro S" first="Stefano" last="Campanaro">Stefano Campanaro</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Biology, University of Padova</institution>, Padova,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="De Pascale, Fabio" sort="De Pascale, Fabio" uniqKey="De Pascale F" first="Fabio" last="De Pascale">Fabio De Pascale</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Biology, University of Padova</institution>, Padova,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Schiavon, Riccardo" sort="Schiavon, Riccardo" uniqKey="Schiavon R" first="Riccardo" last="Schiavon">Riccardo Schiavon</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Biology, University of Padova</institution>, Padova,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Tiarca, Maurizio" sort="Tiarca, Maurizio" uniqKey="Tiarca M" first="Maurizio" last="Tiarca">Maurizio Tiarca</name><affiliation wicri:level="1"><nlm:aff id="a3"><institution>RWL Water/Eurotec WTT, Water Treatment Technologies, S.r.l.</institution>, Padova,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Poletto, Fabio" sort="Poletto, Fabio" uniqKey="Poletto F" first="Fabio" last="Poletto">Fabio Poletto</name><affiliation wicri:level="1"><nlm:aff id="a3"><institution>RWL Water/Eurotec WTT, Water Treatment Technologies, S.r.l.</institution>, Padova,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Concheri, Giuseppe" sort="Concheri, Giuseppe" uniqKey="Concheri G" first="Giuseppe" last="Concheri">Giuseppe Concheri</name><affiliation wicri:level="1"><nlm:aff id="a4"><institution>Department of Agronomy Animals, Food, Natural Resources and Environment, DAFNAE, University of Padova</institution>, Legnaro (Padova)<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Valle, Giorgio" sort="Valle, Giorgio" uniqKey="Valle G" first="Giorgio" last="Valle">Giorgio Valle</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Biology, University of Padova</institution>, Padova,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Squartini, Andrea" sort="Squartini, Andrea" uniqKey="Squartini A" first="Andrea" last="Squartini">Andrea Squartini</name><affiliation wicri:level="1"><nlm:aff id="a4"><institution>Department of Agronomy Animals, Food, Natural Resources and Environment, DAFNAE, University of Padova</institution>, Legnaro (Padova)<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Scientific Reports</title><idno type="eISSN">2045-2322</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The analysis of environmental microbial communities has largely relied on a PCR-dependent amplification of genes entailing species identity as 16S rRNA. This approach is susceptible to biases depending on the level of primer matching in different species. Moreover, possible yet-to-discover taxa whose rRNA could differ enough from known ones would not be revealed. DNA-based methods moreover do not provide information on the actual physiological relevance of each taxon within an environment and are affected by the variable number of rRNA operons in different genomes. To overcome these drawbacks we propose an approach of direct sequencing of 16S ribosomal RNA without any primer- or PCR-dependent step. The method was tested on a microbial community developing in an anammox bioreactor sampled at different time-points. A conventional PCR-based amplicon pyrosequencing was run in parallel. The community resulting from direct rRNA sequencing was highly consistent with the known biochemical processes operative in the reactor. As direct rRNA-seq is based not only on taxon abundance but also on physiological activity, no comparison between its results and those from PCR-based approaches can be applied. The novel principle is in this respect proposed not as an alternative but rather as a complementary methodology in microbial community studies.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Woese, C" uniqKey="Woese C">C. Woese</name></author><author><name sortKey="Fox, G" uniqKey="Fox G">G. Fox</name></author><author><name sortKey="Zablen, L" uniqKey="Zablen L">L. Zablen</name></author><author><name sortKey="Uchida, T" uniqKey="Uchida T">T. Uchida</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Woese, C R" uniqKey="Woese C">C. R. Woese</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rzhetsky, A" uniqKey="Rzhetsky A">A. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Sci Rep</journal-id><journal-id journal-id-type="iso-abbrev">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type="epub">2045-2322</issn><publisher><publisher-name>Nature Publishing Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">27577787</article-id><article-id pub-id-type="pmc">5006002</article-id><article-id pub-id-type="pii">srep32165</article-id><article-id pub-id-type="doi">10.1038/srep32165</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Direct 16S rRNA-seq from bacterial communities: a PCR-independent approach to simultaneously assess microbial diversity and functional activity potential of each taxon</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Rosselli</surname><given-names>Riccardo</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Romoli</surname><given-names>Ottavia</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Vitulo</surname><given-names>Nicola</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Vezzi</surname><given-names>Alessandro</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Campanaro</surname><given-names>Stefano</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>de Pascale</surname><given-names>Fabio</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Schiavon</surname><given-names>Riccardo</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Tiarca</surname><given-names>Maurizio</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Poletto</surname><given-names>Fabio</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Concheri</surname><given-names>Giuseppe</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Valle</surname><given-names>Giorgio</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Squartini</surname><given-names>Andrea</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a4">4</xref></contrib><aff id="a1"><label>1</label><institution>Department of Biology, University of Padova</institution>, Padova,<country>Italy</country></aff><aff id="a2"><label>2</label><institution>Department of Biotechnology, University of Verona</institution>, Verona 37134,<country>Italy</country></aff><aff id="a3"><label>3</label><institution>RWL Water/Eurotec WTT, Water Treatment Technologies, S.r.l.</institution>, Padova,<country>Italy</country></aff><aff id="a4"><label>4</label><institution>Department of Agronomy Animals, Food, Natural Resources and Environment, DAFNAE, University of Padova</institution>, Legnaro (Padova)<country>Italy</country></aff></contrib-group><author-notes><corresp id="c1"><label>a</label><email>squart@unipd.it</email></corresp></author-notes><pub-date pub-type="epub"><day>31</day><month>08</month><year>2016</year></pub-date><pub-date pub-type="collection"><year>2016</year></pub-date><volume>6</volume><elocation-id>32165</elocation-id><history><date date-type="received"><day>05</day><month>02</month><year>2016</year></date><date date-type="accepted"><day>28</day><month>07</month><year>2016</year></date></history><permissions><copyright-statement>Copyright © 2016, The Author(s)</copyright-statement><copyright-year>2016</copyright-year><copyright-holder>The Author(s)</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/4.0/"><pmc-comment>author-paid</pmc-comment>
<license-p>This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link></license-p></license></permissions><abstract><p>The analysis of environmental microbial communities has largely relied on a PCR-dependent amplification of genes entailing species identity as 16S rRNA. This approach is susceptible to biases depending on the level of primer matching in different species. Moreover, possible yet-to-discover taxa whose rRNA could differ enough from known ones would not be revealed. DNA-based methods moreover do not provide information on the actual physiological relevance of each taxon within an environment and are affected by the variable number of rRNA operons in different genomes. To overcome these drawbacks we propose an approach of direct sequencing of 16S ribosomal RNA without any primer- or PCR-dependent step. The method was tested on a microbial community developing in an anammox bioreactor sampled at different time-points. A conventional PCR-based amplicon pyrosequencing was run in parallel. The community resulting from direct rRNA sequencing was highly consistent with the known biochemical processes operative in the reactor. As direct rRNA-seq is based not only on taxon abundance but also on physiological activity, no comparison between its results and those from PCR-based approaches can be applied. The novel principle is in this respect proposed not as an alternative but rather as a complementary methodology in microbial community studies.</p></abstract></article-meta></front><floats-group><fig id="f1"><label>Figure 1</label><caption><title>Outline of the rRNA sequencing and annotation procedure.</title><p>Purple lines: not-uniquely aligned reads. Green lines: uniquely aligned reads. The latter, being specific for a single target within the database, fall on hypervariable regions (vs). (<bold>a</bold>) Paired-end SOLiD RNA-sequencing; (<bold>b</bold>) Double-encoding and read alignments on a 16S-rRNA reference database; (<bold>c</bold>) Selection of candidate subjects. Only subjects covered at least 10% of the total length by unique-aligned reads were selected for the second screening; (<bold>d</bold>) Selection of confirmed subjects. Not-uniquely-aligned reads information was used to confirm the subject. Sequences with at least 50% of total length covered were considered for the annotation; (<bold>e</bold>) Sequences annotation: subjects aligned over encoded and not-encoded (NNN) regions were annotated by CAMERA.</p></caption><graphic xlink:href="srep32165-f1"></graphic></fig><fig id="f2"><label>Figure 2</label><caption><title>Percentages of taxa assigned through database alignment of sequences obtained by 454 (PCR-based) amplicon pyrosequencing (blue bars) and by SOLiD direct rRNA-seq (green bars) for the phyla of the bacteria superkingdom occurring in the anammox bioreactor sludge at the two sampling dates.</title><p>Note the broken scale step to magnify the region below the 6% abundance threshold, separated from the top which shows the 100% value.</p></caption><graphic xlink:href="srep32165-f2"></graphic></fig><fig id="f3"><label>Figure 3</label><caption><title>Percentages of taxa assigned at genus level by the rRNA-seq.</title><p>Day 154 sampling.</p></caption><graphic xlink:href="srep32165-f3"></graphic></fig><fig id="f4"><label>Figure 4</label><caption><title>Percentages of taxa assigned at genus level by the rRNA-seq.</title><p>Day 189 sampling.</p></caption><graphic xlink:href="srep32165-f4"></graphic></fig><table-wrap position="float" id="t1"><label>Table 1</label><caption><title>Percentage of sequences obtained by PCR-based analysis and by rRNA-seq at the two sampling times and ratio of the two values pairs (% by PCR/% by rRNA-seq).</title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col></colgroup><thead valign="bottom"><tr><th rowspan="2" align="left" valign="top" charoff="50">Phylum</th><th colspan="3" align="center" valign="top" charoff="50">Day 154 sampling</th><th colspan="3" align="center" valign="top" charoff="50">Day 189 sampling</th></tr><tr><th align="center" valign="top" charoff="50">% (by PCR)</th><th align="center" valign="top" charoff="50">% (by RNA)</th><th align="center" valign="top" charoff="50">Ratio by PCR/by RNA</th><th align="center" valign="top" charoff="50">% (by PCR)</th><th align="center" valign="top" charoff="50">% (by RNA)</th><th align="center" valign="top" charoff="50">Ratio by PCR/by RNA</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50">Acidobacteria</td><td align="center" valign="top" charoff="50">1.877</td><td align="center" valign="top" charoff="50">0.084</td><td align="center" valign="top" charoff="50">22.256</td><td align="center" valign="top" charoff="50">3.062</td><td align="center" valign="top" charoff="50">0.053</td><td align="center" valign="top" charoff="50">57.819</td></tr><tr><td align="left" valign="top" charoff="50">Actinobacteria</td><td align="center" valign="top" charoff="50">4.530</td><td align="center" valign="top" charoff="50">0.015</td><td align="center" valign="top" charoff="50">308.903</td><td align="center" valign="top" charoff="50">13.604</td><td align="center" valign="top" charoff="50">0.046</td><td align="center" valign="top" charoff="50">298.464</td></tr><tr><td align="left" valign="top" charoff="50">Bacteroidetes</td><td align="center" valign="top" charoff="50">1.359</td><td align="center" valign="top" charoff="50">0.151</td><td align="center" valign="top" charoff="50">9.020</td><td align="center" valign="top" charoff="50">1.590</td><td align="center" valign="top" charoff="50">0.399</td><td align="center" valign="top" charoff="50">3.989</td></tr><tr><td align="left" valign="top" charoff="50">Armatimonadetes</td><td align="center" valign="top" charoff="50">0.302</td><td align="center" valign="top" charoff="50">5.697</td><td align="center" valign="top" charoff="50"><bold>0.053</bold></td><td align="center" valign="top" charoff="50">0.412</td><td align="center" valign="top" charoff="50">6.652</td><td align="center" valign="top" charoff="50"><bold>0.062</bold></td></tr><tr><td align="left" valign="top" charoff="50">Chlorobi</td><td align="center" valign="top" charoff="50">23.598</td><td align="center" valign="top" charoff="50">2.595</td><td align="center" valign="top" charoff="50">9.093</td><td align="center" valign="top" charoff="50">10.984</td><td align="center" valign="top" charoff="50">1.648</td><td align="center" valign="top" charoff="50">6.662</td></tr><tr><td align="left" valign="top" charoff="50">Chloroflexi</td><td align="center" valign="top" charoff="50">2.071</td><td align="center" valign="top" charoff="50">0.867</td><td align="center" valign="top" charoff="50">2.387</td><td align="center" valign="top" charoff="50">1.914</td><td align="center" valign="top" charoff="50">0.978</td><td align="center" valign="top" charoff="50">1.953</td></tr><tr><td align="left" valign="top" charoff="50">Firmicutes</td><td align="center" valign="top" charoff="50">0.669</td><td align="center" valign="top" charoff="50">0.008</td><td align="center" valign="top" charoff="50">81.510</td><td align="center" valign="top" charoff="50">0.383</td><td align="center" valign="top" charoff="50">0.027</td><td align="center" valign="top" charoff="50">14.079</td></tr><tr><td align="left" valign="top" charoff="50">Gemmatimonadetes</td><td align="center" valign="top" charoff="50">4.659</td><td align="center" valign="top" charoff="50">0.206</td><td align="center" valign="top" charoff="50">22.612</td><td align="center" valign="top" charoff="50">6.302</td><td align="center" valign="top" charoff="50">0.177</td><td align="center" valign="top" charoff="50">35.642</td></tr><tr><td align="left" valign="top" charoff="50">Planctomycetes</td><td align="center" valign="top" charoff="50">24.892</td><td align="center" valign="top" charoff="50">87.362</td><td align="center" valign="top" charoff="50"><bold>0.285</bold></td><td align="center" valign="top" charoff="50">14.694</td><td align="center" valign="top" charoff="50">86.459</td><td align="center" valign="top" charoff="50"><bold>0.170</bold></td></tr><tr><td align="left" valign="top" charoff="50">Proteobacteria</td><td align="center" valign="top" charoff="50">34.060</td><td align="center" valign="top" charoff="50">2.705</td><td align="center" valign="top" charoff="50">12.593</td><td align="center" valign="top" charoff="50">43.463</td><td align="center" valign="top" charoff="50">3.301</td><td align="center" valign="top" charoff="50">13.166</td></tr><tr><td align="left" valign="top" charoff="50">Candidate Division BRC1</td><td align="center" valign="top" charoff="50">0.388</td><td align="center" valign="top" charoff="50">0.042</td><td align="center" valign="top" charoff="50">9.233</td><td align="center" valign="top" charoff="50">0.736</td><td align="center" valign="top" charoff="50">0.023</td><td align="center" valign="top" charoff="50">32.872</td></tr><tr><td align="left" valign="top" charoff="50">Candidate Division TM7</td><td align="center" valign="top" charoff="50">0.863</td><td align="center" valign="top" charoff="50">0.006</td><td align="center" valign="top" charoff="50">144.614</td><td align="center" valign="top" charoff="50">0.942</td><td align="center" valign="top" charoff="50">0.030</td><td align="center" valign="top" charoff="50">31.799</td></tr></tbody></table><table-wrap-foot><fn id="t1-fn1"><p>Only phyla for which a minimum of 10 sequences were available are reported in the table. Ratios resulting in values <1 are highlighted in <bold>boldface</bold>.</p></fn></table-wrap-foot></table-wrap></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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