A fungal mock community control for amplicon sequencing experiments

Abstract

Microbial ecology has been profoundly advanced by the ability to profile complex microbial communities by sequencing of marker genes amplified from environmental samples. However, inclusion of appropriate controls is vital to revealing the limitations and biases of this technique. "Mock community" samples, in which the composition and relative abundances of community members are known, are particularly valuable for guiding library preparation and data processing decisions. 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 during library preparation. Increasing the number of PCR cycles elevated rates of chimera formation, and of errors in the final data set. Extension time during PCR had little impact on chimera formation, error rate or observed community structure. Polymerase fidelity impacted error rates significantly. Despite a high error rate, a master mix optimized to minimize amplification bias yielded profiles that were most similar to the true community structure. Bias against particular taxa differed among ITS1 vs. ITS2 loci. Preclustering nearly identical reads substantially reduced error rates, but did not improve similarity to the expected community structure. Inaccuracies in amplicon sequence-based estimates of fungal community structure were associated with amplification bias and size selection processes, as well as variable culling rates among reads from different taxa. In some cases, the numerically dominant taxon was completely absent from final data sets, highlighting the need for further methodological improvements to avoid biased observations of community profiles.

Towards PacBio‐based pan‐eukaryote metabarcoding using full‐length ITS sequences

“…Besides Archaeorhizomycetes, the ITS9munngs + TW13 primer pair tended to overyield Rhizaria and Sordariomycetes and other groups with short sequences but discriminated against Metazoa, Chytridiomycota and Agaricomycetes with relatively long ITS sequences. Thus, our results confirm identification biases in Illumina MiSeq and PacBio RSII platforms against taxa with longer ITS sequences (Bakker ; Tedersoo et al . , ).…”

Optimized metabarcoding with Pacific biosciences enables semi‐quantitative analysis of fungal communities

“…Thus, the interpretation of internal transcribed spacer (ITS) sequencing data as semi‐quantitative (i.e. correctly representing relative abundances of members) in fungal community studies has been questioned based on observations of biases and errors when sequencing artificially assembled (‘mock’) communities (Amend et al ., 2010; Bakker, 2018; Palmer et al ., 2018). Such biases and errors affect perceived differences in composition and diversity among communities, and should be minimized (Frøslev et al ., 2017; Nilsson et al ., 2019).…”

dadasnake, a Snakemake implementation of DADA2 to process amplicon sequencing data for microbial ecology

“…To demonstrate dadasnake's potential to accurately determine community composition and richness, two mock community datasets from Illumina sequencing of bacterial [36] and fungal [37] DNA were analysed. In both cases, the genus-level composition was determined mostly correctly ( Figure 2 a&b; supplementary table 2).…”

“…The ITS2 region of a fungal mock community [37] was amplified using the primers F-ITS4 5-TCCTCCGCTTATTGATATGC [45] and R-fITS7 5-GTGARTCATCGAATCTTTG [46] modified with heterogeneity spacers according to [47]. Amplicon libraries were prepared using the Nextera XT kit (Illumina) and sequenced on an Illumina MiSeq with v.3 chemistry at 2 x 300 bp.…”