Groundtruthing Next-Gen Sequencing for Microbial Ecology-Bia
Details of Research
TitleGroundtruthing Next-Gen Sequencing for Microbial Ecology-Biases and Errors in Community Structure Estimates from PCR Amplicon PyrosequencingAbstractAnalysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa-a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3-V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with >1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure. Â© 2012 Lee et al.
1st AuthorLee, C. AuthorLee, C.Herbold, C.Polson, S.Wommack, K.Williamson, S.McDonald, I.Cary, S.Year2012JournalPLoS ONEVolume7Number9DOI10.1371/journal.pone.0044224URLhttps://www.scopus.com/inward/recor.....0c38643a7cb3292d459cda6beKeywordsRNA 16S, ampliconarticlechimeracommunity structurecontrolled studygene identificationgene librarygene sequencegenetic variabilityhigh throughput sequencingin vitro studylimit of detectionmicrobial communitymolecular cloningnonhumanpolymerase chain reactionpyrosequencingquality controlsystematic errortaxonomy, AlgorithmsBacteriaBase SequenceBias (Epidemiology)BiotaDNA PrimersPolymerase Chain ReactionRNA, Ribosomal, 16SSequence Analysis, DNAStatistics, NonparametricTemperature, Otus
CitationLee, C., Herbold, C., Polson, S., Wommack, K., Williamson, S., McDonald, I. and Cary, S. (2012). Groundtruthing Next-Gen Sequencing for Microbial Ecology-Biases and Errors in Community Structure Estimates from PCR Amplicon Pyrosequencing. PLoS ONE, 7(9)
Groundtruthing Next-Gen Sequencing for Microbial Ecology-Bia Antarctica NZ, accessed 24 May 2022, https://adam.antarcticanz.govt.nz/nodes/view/64224, 10.1371/journal.pone.0044224