A comparative study of microbial diversity and community structure in marine sediments using poly(A) tailing and reverse transcription PCR

Frontiers in Microbiology. 2013;4 DOI 10.3389/fmicb.2013.00160

 

Journal Homepage

Journal Title: Frontiers in Microbiology

ISSN: 1664-302X (Online)

Publisher: Frontiers Media S.A.

LCC Subject Category: Science: Microbiology

Country of publisher: Switzerland

Language of fulltext: English

Full-text formats available: PDF, HTML, ePUB, XML

 

AUTHORS


Tatsuhiko eHoshino (Japan Agency for Marine-Earth Science and Technology)

Tatsuhiko eHoshino (Japan Agency for Marine-Earth Science and Technology (JAMSTEC))

Fumio eInagaki (Japan Agency for Marine-Earth Science and Technology)

Fumio eInagaki (Japan Agency for Marine-Earth Science and Technology (JAMSTEC))

EDITORIAL INFORMATION

Blind peer review

Editorial Board

Instructions for authors

Time From Submission to Publication: 14 weeks

 

Abstract | Full Text

To obtain a better understanding of metabolically active microbial communities, we tested a molecular ecological approach using poly(A) tailing of environmental 16S rRNA, followed by full-length complementary DNA (cDNA) synthesis and sequencing to eliminate potential biases caused by mismatching of PCR primer sequences. The RNA pool tested was extracted from marine sediments of the Yonaguni Knoll IV hydrothermal field in the southern Okinawa Trough. The sequences obtained using the ploy(A) tailing method were compared statistically and phylogenetically with those obtained using conventional reverse transcription-polymerase chain reaction (RT-PCR) with published domain-specific primers. Both methods indicated that Deltaproteobacteria are predominant in sediment (>85% of the total sequence read). The poly(A) tailing method indicated that Desulfobacterales were the predominant deltaproteobacteria, while most of the sequences in libraries constructed using RT-PCR were derived from Desulfuromonadales. This discrepancy may have been due to low coverage of Desulfobacterales by the primers used. A comparison of library diversity indices indicated that the poly(A) tailing method retrieves more phylogenetically diverse sequences from the environment. The four archaeal 16S rRNA sequences that were obtained using the poly(A) tailing method formed deeply branching lineages that were related to Candidatus Parvarchaeum and the Ancient Archaeal Group. These results clearly demonstrate that poly(A) tailing followed by cDNA sequencing is a powerful and less biased molecular ecological approach for the study of metabolically active microbial communities.