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.
Highlights
Fungal disease affects more than 300 million people worldwide, accounting for an estimated 1.5 million deaths annually [1]
The microbiome has been investigated in respiratory disease, with the mycobiome more recently emerging as an important area of research [9,28,29]
In this study we sought to define the utility of several primer sets that target the ITS1 or ITS2 regions of the fungal 18S–28S intergenic regions, in the characterization of the human airway mycobiome
Summary
Fungal disease affects more than 300 million people worldwide, accounting for an estimated 1.5 million deaths annually [1]. While phagocytic and mucociliary clearance are employed effectively in healthy non-immunocompromised individuals, those with chronic respiratory diseases such as chronic obstructive pulmonary disease (COPD) or bronchiectasis have anatomically abnormal airways and dysfunctional immunity. This results in higher risks of fungal acquisition, colonization, and potential complications [2]. Targeted amplicon sequencing is used in most published work focused on the mycobiome This contrasts shotgun sequencing, which is hindered by the greater relative abundance of bacterial DNA to that of fungi in clinical samples [10]. While the internally transcribed spacer region (ITS) is recognized as a universal taxonomic barcode for fungi, appraisal of different ITS subregions and the influence of DNA extraction methods have not been comprehensively examined using respiratory specimens, making it challenging to compare results between existing respiratory mycobiome studies, both between disease states and in different patient cohorts [12,13,14,15]
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