Experimental factors affecting PCR-based estimates of microbial species richness and evenness

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Pyrosequencing of 16S rRNA gene amplicons for microbial community profiling can, for equivalent costs, yield greater than two orders of magnitude more sensitivity than traditional PCR-cloning and Sanger sequencing. With this increased sensitivity and the ability to analyze multiple samples in parallel, it has become possible to evaluate several technical aspects of PCRbased community structure profiling methods. We tested the effect of amplicon length and primer pair on estimates of species richness number of species and evenness relative abundance of species by assessing the potentially tractable microbial community residing in the termite hindgut. Two regions of the 16S rRNA gene ... continued below

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Engelbrektson, Anna; Kunin, Victor; Wrighton, Kelly C.; Zvenigorodsky, Natasha; Chen, Feng; Ochman, Howard et al. December 1, 2009.

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Pyrosequencing of 16S rRNA gene amplicons for microbial community profiling can, for equivalent costs, yield greater than two orders of magnitude more sensitivity than traditional PCR-cloning and Sanger sequencing. With this increased sensitivity and the ability to analyze multiple samples in parallel, it has become possible to evaluate several technical aspects of PCRbased community structure profiling methods. We tested the effect of amplicon length and primer pair on estimates of species richness number of species and evenness relative abundance of species by assessing the potentially tractable microbial community residing in the termite hindgut. Two regions of the 16S rRNA gene were sequenced from one of two common priming sites, spanning the V1-V2 or V8 regions, using amplicons ranging n length from 352 to 1443 bp. Our results demonstrate that both amplicon length and primer pair markedly influence estimates of richness and evenness. However, estimates of species evenness are consistent among different primer pairs targeting the same region. These results highlight the importance of experimental methodology when comparing diversity estimates across communities.

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  • Journal Name: ISME Journal

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  • Report No.: LBNL-2917E
  • Grant Number: DE-AC02-05CH11231
  • Office of Scientific & Technical Information Report Number: 982046
  • Archival Resource Key: ark:/67531/metadc1015007

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  • December 1, 2009

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  • Oct. 14, 2017, 8:36 a.m.

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  • Oct. 17, 2017, 8:14 p.m.

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Engelbrektson, Anna; Kunin, Victor; Wrighton, Kelly C.; Zvenigorodsky, Natasha; Chen, Feng; Ochman, Howard et al. Experimental factors affecting PCR-based estimates of microbial species richness and evenness, article, December 1, 2009; Berkeley, California. (digital.library.unt.edu/ark:/67531/metadc1015007/: accessed December 16, 2017), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.