Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 8/3/2017
Publication Date: N/A
Technical Abstract: The field of microbial ecology has been profoundly advanced by the ability to profile the composition of complex microbial communities by means of high throughput amplicon sequencing of marker genes amplified directly from environmental genomic DNA extracts. However, it has become increasingly clear that amplicon sequencing techniques carry their own limitations and biases. Appropriate controls are required to make these biases visible, and will protect against erroneous or naïve conclusions. In particular, the inclusion of ‘mock community’ samples, in which the composition and relative abundances of community members are known in advance, is particularly valuable. I generated a set of three mock communities using 19 different fungal taxa, and demonstrate their utility by contrasting amplicon sequencing data obtained for the same communities under modifications to PCR conditions and the polymerase enzyme used during library preparation. A larger number of PCR cycles during amplicon generation increased rates of chimera formation, and of errors in the final dataset. Increasing duration of PCR extension time increased chimera formation rate slightly, but did not impact final error rate. A high-fidelity Phusion polymerase and master mix generated data with a significantly lower error rate compared to a standard Taq polymerase. The ability of preclustering (binning together sequences having only a set number of differences) to improve accuracy varied among primer sets and marker gene loci. In general, tinkering with PCR conditions could not alleviate fundamental inaccuracies in estimates of community structure, which were associated with amplification bias and size selection processes. Despite a high error rate, a polymerase and master mix optimized to minimize amplification bias (a pre-amplification master mix geared toward gene expression experiments) best captured fungal community structure, in certain cases. A requirement for inclusion of mock community controls would improve the quality of current amplicon sequencing studies, by forcing researchers to confront the sometimes severe limitations of the method, and would facilitate development of improved methods for accurately profiling the community structure of environmental microbiomes.