Characterizing the Human Mycobiota: A Comparison of Small Subunit rRNA, ITS1, ITS2, and Large Subunit rRNA Genomic Targets - PubMed (original) (raw)
Characterizing the Human Mycobiota: A Comparison of Small Subunit rRNA, ITS1, ITS2, and Large Subunit rRNA Genomic Targets
Michael Hoggard et al. Front Microbiol. 2018.
Abstract
Interest in the human microbiome has increased dramatically in the last decade. However, much of this research has focused on bacteria, while the composition and roles of their fungal counterparts remain less understood. Furthermore, a variety of methodological approaches have been applied, and the comparability between studies is unclear. This study compared four primer pairs targeting the small subunit (SSU) rRNA (18S), ITS1, ITS2, and large subunit (LSU) rRNA (26S) genomic regions for their ability to accurately characterize fungal communities typical of the human mycobiota. All four target regions of 21 individual fungal mock community taxa were capable of being amplified adequately and sequenced. Mixed mock community analyses revealed marked variability in the ability of each primer pair to accurately characterize a complex community. ITS target regions outperformed LSU and SSU. Of the ITS regions, ITS1 failed to generate sequences for Yarrowia lipolytica and all three Malassezia species when in a mixed community. These findings were further supported in studies of human sinonasal and mouse fecal samples. Based on these analyses, previous studies using ITS1, SSU, or LSU markers may omit key taxa that are identified by the ITS2 marker. Of methods commonly used in human mycobiota studies to date, we recommend selection of the ITS2 marker. Further investigation of more recently developed fungal primer options will be essential to ultimately determine the optimal methodological approach by which future human mycobiota studies ought to be standardized.
Keywords: fungi; human microbiome; internal transcribed spacer; microbiota; mycobiota; next-generation sequencing.
Figures
FIGURE 1
Methodological overview. (A) The fungal ribosomal cistron, together with the primers and target regions assessed in this study. ITS, internal transcribed spacer; SSU, small subunit; LSU, large subunit. (B) Samples were collected from 21 fungal isolates, human upper-respiratory tract (sinonasal swab) (n = 10), and mouse gut (fecal) material (n = 10). All samples were amplified using primer pairs for four target genomic regions and sequenced on the Illumina MiSeq platform to compare the ability of each to accurately characterize the fungal communities in the samples. Readily available reference databases were also compared to assess the accuracy of taxonomic assignments of each of the sequence data sets.
FIGURE 2
The human mycobiota mock community. (A) Full list of species included in the human mycobiota mock community assembled for this study (phylum; class; genus; and species). All taxa were sequenced individually and as a mixed mock community. (B) Accuracy of taxonomic assignments for each of the 21 taxa (sequenced individually) and four target regions based on UNITE (ITS), RDP (ITS, LSU), and SILVA (SSU, LSU) reference databases. (C) Relative proportion of sequences for each species when amplified and sequenced as a mixed mock community, color-coded by whether sequences for each were over-represented (red) or under-represented (blue) compared to the expected (even) distribution (expected = 4.7% relative abundance for each species). ∗A. brasiliensis and A. niger shared the same ZOTU for each of ITS1, ITS2, and LSU (i.e., had identical sequences), and C. albicans and C. parapsilosis shared the same ZOTU for SSU. ∗∗A. brasiliensis was split across numerous ZOTUs for SSU sequences and had mixed classification, making relative abundance estimates not possible for SSU.
FIGURE 3
Human mycobiota mock community sequence data summary. (A) nMDS plot of replicates for each of the four target genomic regions [ITS1, ITS2, SSU (18S), LSU (26S)] and taxonomic reference databases (UNITE, RDP, SILVA) compared to the expected community structure (even distribution: “Mock21_Expected”), based on weighted Bray–Curtis dissimilarity. (B) Taxonomic summary plot for each of the replicates compared with the expected community structure, with samples ordered by average-linkage hierarchical clustering (represented by the dendrogram above). Dashed line boxes indicate partitioning of the samples into five clusters based on hierarchical clustering.
FIGURE 4
Human upper respiratory and mouse gut ITS data summaries. Taxonomic summary plots comparing ITS1 and ITS2 sequence data for (A) human sinonasal (n = 10) and (B) mouse fecal samples (n = 10), with taxonomic assignments conducted using the RDP ITS reference database. Dashed-line bars indicate samples that did not amplify sufficiently and/or meet the sequence subsampling threshold of 1000 reads per sample.
FIGURE 5
ITS1 and ITS2 fungal community coverage. Venn diagrams representing the number of unique taxonomic assignments identified by ITS1 only (yellow), ITS2 only (blue), or both (green), for (A) human sinonasal, and (B) mouse fecal samples. Taxonomic assignments were based on RDP ITS reference database. Full taxonomy lists used to generate the Venn diagrams are provided in Supplementary Figure 2.
References
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