Accuracy of the high-throughput amplicon sequencing to identify species within the genus Aspergillus

• Accuracy of high-throughput sequencing to identify Aspergillus species was examined.
• 300-bp sequencing of ITS1, BenA, and CaM was accurate at least at section rank.
• Ambiguities were observed at species rank due to cross detection of sibling taxa.
• In silico analysis predicted 107–187 species were identifiable down to species rank.
• Detection can be taxonomically biased depending on the DNA markers and/or primers.

This study characterized the accuracy of high-throughput amplicon sequencing to identify species within the genus Aspergillus. To this end, we sequenced the internal transcribed spacer 1 (ITS1), β-tubulin (BenA), and calmodulin (CaM) gene encoding sequences as DNA markers from eight reference Aspergillus strains with known identities using 300-bp sequencing on the Illumina MiSeq platform, and compared them with the BLASTn outputs. The identifications with the sequences longer than 250 bp were accurate at the section rank, with some ambiguities observed at the species rank due to mostly cross detection of sibling species. Additionally, in silico analysis was performed to predict the identification accuracy for all species in the genus Aspergillus, where 107, 210, and 187 species were predicted to be identifiable down to the species rank based on ITS1, BenA, and CaM, respectively. Finally, air filter samples were analysed to quantify the relative abundances of Aspergillus species in outdoor air. The results were reproducible across biological duplicates both at the species and section ranks, but not strongly correlated between ITS1 and BenA, suggesting the Aspergillus detection can be taxonomically biased depending on the selection of the DNA markers and/or primers.