Polymerase matters: non-proofreading enzymes inflate fungal community richness estimates by up to 15 %

Abstract

a b s t r a c tRare taxa overwhelm metabarcoding data generated using next-generation sequencing (NGS). Low frequency Operational Taxonomic Units (OTUs) may be artifacts generated by PCR-amplification errors resulting from polymerase mispairing. We analyzed two Internal Transcribed Spacer 2 (ITS2) MiSeq libraries generated with proofreading (ThermoScientific Phusion Ò ) and non-proofreading (ThermoScientific Phire Ò ) polymerases from the same MiSeq reaction, the same samples, using the same DNA tags, and with two different clustering methods to evaluate the effect of polymerase and clustering tool choices on the estimates of richness, diversity and community composition. Our data show that, while the overall communities are comparable, OTU richness is exaggerated by the use of the non-proofreading polymeraseeup to 15 % depending on the clustering method, and on the threshold of low frequency OTU removal. The overestimation of richness also consistently led to underestimation of community evenness, a result of increase in the low frequency OTUs. Stringent thresholds of eliminating the rare reads remedy this issue; exclusion of reads that occurred 10 times reduced overestimated OTU numbers to <0.3 %. As a result of these findings, we strongly recommend the use of proofreading polymerases to improve the data integrity as well as the use of stringent culling thresholds for rare sequences to minimize overestimation of community richness. ª

PacBio metabarcoding of Fungi and other eukaryotes: errors, biases and perspectives

“…Our PacBio and Illumina substitution profiles match closely to the Taq polymerase error profile (Potapov & Ong, ), raising concern that the relatively sensitive proofreading enzyme may have been inactive during the amplification of our fungal and soil DNA samples. The choice and testing of polymerase and optimal amplification conditions are critical for the minimization of random errors in HTS data (Oliver et al ., ; D'Amore et al ., ; Gohl et al ., ; Potapov & Ong, ).…”

Short‐term direct contact with soil and plant materials leads to an immediate increase in diversity of skin microbiota

“…Sequences classified to Chloroplast, Mitochondria, unknown, Archaea, and Eukaryota were removed from the analyses. Rare OTUs that were represented by 10 or fewer sequences in the whole data were removed as suggested by Oliver, Brown, Callaham, and Jumpponen (2015). For each OTU the number of sequences found in negative controls were subtracted from each sample.…”

Don't put all your eggs in one basket: a cost‐effective and powerful method to optimize primer choice for rRNA environmental community analyses using the Fluidigm Access Array

“…Huse et al 2010;Kunin et al 2010;Tedersoo et al 2010;Brown et al 2015a,b). These post-sequencing technical investigations are crucial to environmental sequencing efforts, but equally important are presequencing considerations such as proper experimental design (Lindahl et al 2013) and amplicon generation methods (see Bazzicalupo et al 2013;Blaalid et al 2013;Oliver et al 2015).…”

“…Although methodological obstacles in community sequencing are numerous including polymerase choice (Oliver et al . ), computational limitations (Schloss et al . ) and algorithm choice (Schmidt et al .…”

“…This ability to directly interrogate microbial communities using locus-targeted sequencing of environmental DNA without relying on time-consuming and potentially biased culturing techniques has transformed our understanding of the hyperdiversity and basic ecology of microbial communities (Sogin et al 2006;Jumpponen & Jones 2009). Although methodological obstacles in community sequencing are numerous including polymerase choice (Oliver et al 2015), computational limitations (Schloss et al 2009) and algorithm choice (Schmidt et al 2015), one important aspect of experimental design remains insufficiently examined: the choice of the most efficient primers for amplification of environmental DNA.…”