Improved selection of internal transcribed spacer-specific primers enables quantitative, ultra-high-throughput profiling of fungal communities

Abstract

Ultra-high-throughput sequencing (HTS) of fungal communities has been restricted by short read lengths and primer amplification bias, slowing the adoption of newer sequencing technologies to fungal community profiling. To address these issues, we evaluated the performance of several common internal transcribed spacer (ITS) primers and designed a novel primer set and work flow for simultaneous quantification and species-level interrogation of fungal consortia. Primer comparison and validation were predicted in silico and by sequencing a "mock community" of mixed yeast species to explore the challenges of amplicon length and amplification bias for reconstructing defined yeast community structures. The amplicon size and distribution of this primer set are smaller than for all preexisting ITS primer sets, maximizing sequencing coverage of hypervariable ITS domains by very-short-amplicon, high-throughput sequencing platforms. This feature also enables the optional integration of quantitative PCR (qPCR) directly into the HTS preparatory work flow by substituting qPCR with these primers for standard PCR, yielding quantification of individual community members. The complete work flow described here, utilizing any of the qualified primer sets evaluated, can rapidly profile mixed fungal communities and capably reconstructed well-characterized beer and wine fermentation fungal communities.

Fig 1

Predicted subphylum coverage of UNITE database ITS sequences by primers located in terminal SSU (A), 5.8S (B), and terminal LSU (C) loci of the rRNA operon. Abbreviations for subphyla: Agar., Agaricomycotina; Ent., Entomophthoromycotina; In., incertae sedis; Kick., Kickxellomycotina; Muc., Mucoromycotina; Pezz., Pezizomycotina; Pucc., Pucciniomycotina; Sacc., Saccharomycotina; Taph., Taphrinomycotina; Ust., Ustilaginomycotina.

Fig 2

Average predicted taxonomic classification accuracy of selected primer sets against UNITE database sequences across all phyla. Predicted primer hits against database were trimmed to 150 bp (A) or 250 bp (B) and classified using BLAST. Accuracy represents the fraction of scores matching the correct taxon at each taxonomic level. Primer sets represent intended pairs targeting the ITS1 or ITS2.

Fig 3

Taxonomic distribution of OTUs detected in a single yeast mock community reveals differential bias of ITS primer sets. Shown is the HTS community structure of a defined yeast mock community amplified with five different paired ITS primers. E, expected.

Fig 4

Comparison of ITS sequencing to ITS-TRFLP of wine fungal communities. Botrytized wine fermentations were analyzed with BITS-B58S3 HTS (A) and ITS1-ITS4 TRFLP (B). Panel B was adapted from reference 2 with permission.