Optimisation and Benchmarking of Targeted Amplicon Sequencing for Mycobiome Analysis of Respiratory Specimens

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.

The Mycobiome in Health and Disease: Emerging Concepts, Methodologies and Challenges

“…In the following sections, we aim to describe our current understanding of the mycobiome in the context of recent NGS advances and the methodological challenges and barriers that exist in terms of its clinical implementation. However, recent work from our group, using spontaneous induced sputum, has demonstrated more consistent amplification of the ITS region from samples subjected to mechanical disruption as compared to enzymatic lysis [40]. This was observed despite higher overall DNA yields achieved by enzymatic lysis suggesting preferential liberation of fungal DNA by mechanical lysis [40].…”

“…However, recent work from our group, using spontaneous induced sputum, has demonstrated more consistent amplification of the ITS region from samples subjected to mechanical disruption as compared to enzymatic lysis [40]. This was observed despite higher overall DNA yields achieved by enzymatic lysis suggesting preferential liberation of fungal DNA by mechanical lysis [40]. Extraction methods are critical and must be applied consistently and carefully both within and between mycobiome studies.…”

“…However, demonstration of its utility within the medical diagnostic setting is currently limited. Low abundance of fungi in relation to the overall microbial community (B 0.1%) poses challenges to their detection via metagenomic sequencing in human samples, necessitating targeted amplicon sequencing approaches, which are still in their infancy [29,39,40]. Further refinement and standardisation of reproducible wet-lab and bioinformatic workflows coupled to automated and scalable user-friendly reporting systems remain key challenges for the field, which unless addressed, will continue to impede widespread adoption and translation into clinical practice [41][42][43].…”

“…ITS1F and ITS2) have been used for decades in several large-scale microbiome projects, to date, it is still unclear which ITS fragment is optimal. Recent findings demonstrate that these primers suffer from primer bias, amplification bias and fail to accurately profile mock communities analysed, leading to inconsistent representation of synthetic fungal communities of known taxonomic abundance [40,74,[78][79][80][81][82]. Based on these observations, Nilsson and colleagues provide a list of fungus specific, HTS-oriented primers for amplification of ITS region, and recommend targeting the ITS2 sub-region by using degenerate forward primers gITS7ngs and the reverse primer ITS4ng owing to their superior coverage of the fungal kingdom [60].…”

Evaluation of Droplet Digital Polymerase Chain Reaction (ddPCR) for the Absolute Quantification of Aspergillus species in the Human Airway

“…Having identified that ddPCR demonstrates greater sensitivity for the detection of A. fumigatus and A. terreus in earlier experiments, we next evaluated differences in detection and quantification ability using clinical specimens from healthy and diseased individuals. We prospectively recruited n = 20 individuals (n = 4 non-diseased (healthy), n = 8 with COPD and n = 8 with bronchiectasis), the latter diseased groups with known and previously detectable Aspergillus species based on 18S ITS mycobiome sequencing from our ongoing or previously published works [1,3,15]. While we did not detect any A. fumigatus or A. terreus in non-diseased (healthy) individuals, all patients with COPD and bronchiectasis demonstrated detectable A. fumigatus and/or A. terreus in their airway specimens by qPCR and/or ddPCR (Figure 4).…”