A comparative study of microbial diversity and community structure in marine sediments using poly(A) tailing and reverse transcription-PCR.
A comparative study of microbial diversity and community structure in marine sediments using poly(A) tailing and reverse transcription-PCR.
复制标题
DOI：
10.3389/fmicb.2013.00160
复制
发表时间：
2013
影响因子：
5.2
通讯作者：
Inagaki F
中科院分区：
文献类型：
--
作者：
Hoshino T;Inagaki F
为了更好地了解代谢活跃的微生物群落，我们测试了一种分子生态学方法，使用环境16 S rRNA的poly（A）加尾，然后进行全长互补DNA（cDNA）合成和测序，以消除聚合酶链反应（PCR）引物序列错配引起的潜在偏差。RNA池测试提取自海洋沉积物的与那国穹IV热液田南部冲绳海槽。使用poly（A）加尾方法获得的序列进行了统计学和遗传学比较与使用常规的逆转录-PCR（RT-PCR）与公布的域特异性引物获得的序列。两种方法均表明三角形变形菌在沉积物中占优势（>85%的总序列读数）。poly（A）加尾法表明脱硫菌目是优势的DeltaProteobacteria，而利用RT-PCR构建的文库中的大部分序列来自脱硫单胞菌目。这种差异可能是由于所用引物对脱硫菌目的覆盖率较低。文库多样性指数的比较表明，poly（A）加尾方法从环境中检索到更多的遗传多样性序列。使用poly（A）加尾方法获得的四个古菌16 S rRNA序列形成了与古生菌和古生菌群相关的深分支谱系。这些结果清楚地表明，聚（A）加尾后的cDNA测序是一个强大的和偏见较小的分子生态学方法的代谢活性微生物群落的研究。
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 polymerase chain reaction (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 poly(A) tailing method were compared statistically and phylogenetically with those obtained using conventional reverse transcription-PCR (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.
登录
查看更多内容
影响因子：
14.9
作者：
Klindworth A;Pruesse E;Schweer T;Peplies J;Quast C;Horn M;Glöckner FO
通讯作者：
Glöckner FO
影响因子：
14.9
作者：
Ludwig, W;Strunk, O;Schleifer, KH
通讯作者：
Schleifer, KH
影响因子：
4.4
作者：
HICKS, RE;AMANN, RI;STAHL, DA
通讯作者：
STAHL, DA
影响因子：
4.4
作者：
Loy, A;Lehner, A;Wagner, M
通讯作者：
Wagner, M
影响因子：
5.2
作者：
Hoshino T;Morono Y;Terada T;Imachi H;Ferdelman TG;Inagaki F
通讯作者：
Inagaki F