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Title: Optimisation and benchmarking of targeted amplicon sequencing for mycobiome analysis of respiratory specimens
Authors: Nur A'tikah Mohamed Ali
Aogáin, Micheál Mac
Morales, Raika Francesca
Tiew, Pei Yee
Chotirmall, Sanjay Haresh
Keywords: Science::Medicine
Issue Date: 2019
Source: Nur A'tikah Mohamed Ali, Aogáin, M. M., Morales, R. F., Tiew, P. Y., & Chotirmall, S. H. (2019). Optimisation and benchmarking of targeted amplicon sequencing for mycobiome analysis of respiratory specimens. International Journal of Molecular Sciences, 20(20), 4991-. doi:10.3390/ijms20204991
Journal: International Journal of Molecular Sciences
Abstract: (1) Background: Firm consensus has yet to be established in relation to taxonomic classification and primer choice in targeted amplicon sequencing of the mycobiome. While the nuclear ribosomal internal transcribed spacer (ITS) region are recognized as the formal fungal taxonomic barcode, appraisal of different ITS sub-regions and the influence of DNA extraction methods have not been comprehensively undertaken using human respiratory specimens. (2) Methods: We performed ITS analysis of respiratory (sputum) samples by assessing (a) the effect of alternate DNA extraction techniques and (b) an evaluation of four different ITS primer pairs (ITS1F and ITS2; ITS1-30F and ITS1-217R; gITS7ngs and ITS4ng; and Fseq and Rseq) on the mycobiome profiles generated for mock fungal communities and their respective clinical (airway) specimens. (3) Results: Primer pairs varied in their resulting ITS mycobiome profiles, suggesting that particular pairs may be more relevant for analysis of respiratory samples compared to others. Assessment of DNA extraction methods highlighted lower final DNA concentrations achieved by mechanical disruption compared to enzymatic lysis. However, despite lower yields, DNA liberated by mechanical lysis more readily yielded ITS bands with highest success in combination with the Fseq and Rseq primers. (4) Conclusion: Choice of extraction method, primers used, and sequencing approach are all important considerations in sequencing the mycobiome and should be tailored to sample type. A standardization of approach to mycobiome studies using respiratory specimens will permit more reliable comparisons between studies and improve our understanding of the role of fungi in the human airway.
ISSN: 1661-6596
DOI: 10.3390/ijms20204991
Rights: © 2019 The Authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (
Fulltext Permission: open
Fulltext Availability: With Fulltext
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