Novel ITS1 Fungal Primers for Characterization of the Mycobiome

Abstract

Studies of the human microbiome frequently omit characterization of fungal communities (the mycobiome), which limits our ability to investigate how fungal communities influence human health. The internal transcribed spacer 1 (ITS1) region of the eukaryotic ribosomal cluster has features allowing for wide taxonomic coverage and has been recognized as a suitable barcode region for species-level identification of fungal organisms. We developed custom ITS1 primer sets using iterative alignment refinement. Primer performance was evaluated using in silico testing and experimental testing of fungal cultures and human samples. Using an expanded novel reference database, SIS (18S-ITS1-5.8S), the newly designed primers showed an average in silico taxonomic coverage of 79.9% ± 7.1% compared to a coverage of 44.6% ± 13.2% using previously published primers (P = 0.05). The newly described primer sets recovered an average of 21,830 ± 225 fungal reads from fungal isolate culture samples, whereas the previously published primers had an average of 3,305 ± 1,621 reads (P = 0.03). Of note was an increase in the taxonomic coverage of the Candida genus, which went from a mean coverage of 59.5% ± 13% to 100.0% ± 0.0% (P = 0.0015) comparing the previously described primers to the new primers, respectively. The newly developed ITS1 primer sets significantly improve general taxonomic coverage of fungal communities infecting humans and increased read depth by an order of magnitude over the best-performing published primer set tested. The overall best-performing primer pair in terms of taxonomic coverage and read recovery, ITS1-30F/ITS1-217R, will aid in advancing research in the area of the human mycobiome. IMPORTANCE The mycobiome constitutes all the fungal organisms within an environment or biological niche. The fungi are eukaryotes, are extremely heterogeneous, and include yeasts and molds that colonize humans as part of the microbiome. In addition, fungi can also infect humans and cause disease. Characterization of the bacterial component of the microbiome was revolutionized by 16S rRNA gene fragment amplification, next-generation sequencing technologies, and bioinformatics pipelines. Characterization of the mycobiome has often not been included in microbiome studies because of limitations in amplification systems. This report revisited the selection of PCR primers that amplify the fungal ITS1 region. We have identified primers with superior identification of fungi present in the database. We have compared the new primer sets against those previously used in the literature and show a significant improvement in read count and taxon identification. These primers should facilitate the study of fungi in human physiology and disease states.

Keywords: ITS1; fungi; mycobiome; oral; primer design; yeast.

Figures

FIG 1

ITS rRNA gene locus. Schematic of the eukaryotic ribosomal gene cluster. The SILVA database contains sequences of the 18S gene, while the UNITE database contains sequences from the ITS1-5.8S-ITS2-25S rRNA gene cluster (not to scale). For development of our custom primers, we created a merged SILVA and UNITE database to simulate the 18S-ITS1-5.8S region. A 250-bp region at the 3′ end of the 18S gene was individually isolated when designing the forward primers.

FIG 2

In silico coverage across fungal phyla. Predicted taxonomic coverage was assessed using PrimerProspector. (A) The forward literature fungal primers ITS1 (L), ITS1-F (L), and ITS5 (L) had significantly lower overall taxonomic coverage than the (B) newly created forward primers ITS1-27F (N), ITS1-30F (N), ITS1-34F (N), and ITS1-48F (N). The custom-designed reverse primer ITS1-217R (N) and the published reverse primer ITS2 (L) both demonstrated high predicted taxonomic coverage across the phyla.

FIG 3

In silico taxon coverage. The average in silico taxonomic coverage estimates obtained with PrimerProspector for the selected forward primers from the literature [designated “(L)”] and newly designed forward primers [designated “(N)”]. The published ITS1 (L) primer was recommended by UNITE and is therefore shown in this figure despite the low coverage performance. Coverage estimates are based on 5,789 simulated species in the SIS database. Coverage estimates are based on the default alignment criteria of PrimerProspector.

FIG 4

Experimental NGS read recovery. Average read recovery for the selected newly designed [designated “(N)”] and published forward primers from the literature [designated “(L)”] is shown at the top of each colored bar. Error bars show the standard error of the mean. The names of the primer pairs are indicated on the x axis.

FIG 5

Shannon rarefaction analysis. Shown is Shannon alpha rarefaction analysis across three body sites for seven clinical samples using four newly designed primer pairs. Amplified samples were evaluated at depths of 1, 10, 100, 500, and 1,000 reads, with 100 replicates at each subsampling depth. Results for each primer pair with the samples were averaged and plotted by anatomic site. The primer pairs associated with each colored line are indicated in the key to the right of the figure.