Groundtruthing Next-Gen Sequencing for Microbial Ecology–Biases and Errors in Community Structure Estimates from PCR Amplicon Pyrosequencing

Charles K Lee,S Craig Cary,Shawn W Polson,Shannon J Williamson,Ian R Mcdonald,K Eric Wommack,Craig W Herbold

doi:10.1371/journal.pone.0044224

Charles K Lee, S Craig Cary + Show 5 more

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https://doi.org/10.1371/journal.pone.0044224

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Abstract

Analysis 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.

Highlights

High-throughput pyrosequencing of PCR amplicons has emerged as a valuable technique in microbial ecology and revealed, in unprecedented detail, the microbial diversities found in various marine and terrestrial environments [1,2,3,4,5,6,7,8,9] and the human microbiome [10,11,12,13]
In the context of genomic sequencing, low consensus error rates are achieved through sequence assembly; for PCR amplicons, redundancy is indistinguishable from abundance, and the high error rates associated with individual reads contribute to overestimation of diversity since erroneous reads manifest themselves as less abundant but closely related operational taxonomic unit (OTU) [15]
PCR-independent in vitro-simulated communities (iv-SCs) V3V4P and V6P tested the neutrality of 454-Ti pyrosequencing as they were constructed using individually generated amplicons pooled at known abundances (Table 1)

Summary

Introduction

High-throughput pyrosequencing of PCR amplicons has emerged as a valuable technique in microbial ecology and revealed, in unprecedented detail, the microbial diversities found in various marine and terrestrial environments [1,2,3,4,5,6,7,8,9] and the human microbiome [10,11,12,13] The power of this approach lies in the read depth achieved, where tens to hundreds of thousands of individual sequencing reads are simultaneously generated and used to estimate the composition and abundance of microbial operational taxonomic units (OTUs) in a given community. More stringent approaches have been developed that allow the abundances of error-containing reads to be counted toward those of the more abundant, supposedly error-free, reads from which they arose [16,17,18,19,20,21]

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