Porcine fungal mock community analyses: Implications for mycobiome investigations

Authors: ARKEN, ANN - US Department Of Agriculture (USDA); Foster Frey, Juli; CARRILLO, NORA - US Department Of Agriculture (USDA); DIKE, NNEKA - US Department Of Agriculture (USDA); ONYEACHONAMM, OGECHUWKU - US Department Of Agriculture (USDA); RIVERA, DANIELA - US Department Of Agriculture (USDA); Davies, Cary; Summers, Katie

Submitted to: Frontiers in Cellular and Infection Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/16/2023
Publication Date: 2/8/2023
Citation: Arken, A.M., Foster Frey, J.A., Carrillo, N.I., Dike, N.I., Onyeachonamm, O., Rivera, D.R., Davies, C.L., Summers, K.L. 2023. Porcine fungal mock community analyses: Implications for mycobiome investigations. Frontiers in Cellular and Infection Microbiology. https://doi.org/10.3389/fcimb.2023.928353.
DOI: https://doi.org/10.3389/fcimb.2023.928353

Interpretive Summary: The gut microbiome is an integral partner in host health and plays a role in immune development, altered nutrition, and pathogen prevention. The mycobiome (fungal microbiome) is considered part of the rare biosphere but is still a critical component in health. The low relative abundance of the gut mycobiota and difficulties in fungal taxonomical identification present a variety of methodological challenges in mycobiome characterization, including incomplete or biased community sequencing data. Scientists at the Beltsville Agricultural Research Service, Beltsville, Maryland created mock communities from 5 common fungal isolates found in weanling piglet feces and assessed the potential biases in mycobiome identification based on DNA input, primer target choice, complexity of the community, and the databases utilized for analysis of Illumina MiSeq datasets. A commercial fungal mock community and piglet fecal samples were included in the analysis for comparison. Results obtained using the UNITE and SILVA databases were compared to taxonomic profiles of mock community isolates and database inaccuracies were evident. Primer target region was a critical determinant of species output as well with biases seen based on gene copy number and primer design. Overall, the combination of ITS2 primers and the UNITE database led to the most accurate identifications for piglet fecal samples, the piglet mock community, and the commercial mock community. This study underscores the importance of experimental design and preliminary studies to assess primer combinations and database choice for the mycobiome sample of interest.

Technical Abstract: The gut microbiome is an integral partner in host health and plays a role in immune development, altered nutrition, and pathogen prevention. The mycobiome (fungal microbiome) is considered part of the rare biosphere but is still a critical component in health. The low relative abundance of the gut mycobiota and difficulties in fungal taxonomical identification present a variety of methodological challenges in mycobiome characterization, including incomplete or biased community sequencing data. Although next generation sequencing has rapidly improved our understanding of the microbiome, mycobiome studies necessitate careful assessment of techniques including DNA isolation method, primer design and choice, polymerase selection, sequencing platform, and reference database composition. To better understand the impacts of these factors, we created mock communities from 5 common fungal isolates found in weanling piglet feces and assessed the potential biases in mycobiome identification based on DNA input, primer target choice, complexity of the community, and the databases utilized for analysis of Illumina MiSeq datasets. We also included a commercial fungal mock community and piglet fecal samples in the analysis for comparison. Primer target region was a critical determinant of species output. In fecal samples, 18S and ITS1 libraries displayed the most non-fungal amplicons with an average of 25.7% and 38.1%, respectively, while ITS2 libraries resulted in 7.5%. One fungal isolate, Lichtheimia corymbifera, was not amplified by ITS1 and ITS2 primers, despite being a common thermophilic fungus, suggesting previous studies may be underestimating its prevalence. Estimated gene copy numbers were calculated for the 18S, ITS1, and ITS2 regions of each isolate to determine potential amplification bias. Kazachstania slooffiae displayed the most stable copy numbers (83-85) while L. corymbifera displayed significant variability (90-144) across gene regions. Results obtained using the UNITE and SILVA databases were compared to taxonomic profiles of mock community isolates and database inaccuracies were evident. Overall, the combination of ITS2 primers and the UNITE database led to the most accurate identifications for piglet fecal samples, the piglet mock community, and the commercial mock community. This study underscores the importance of preliminary studies to assess primer combinations and database choice for the mycobiome sample of interest.