Tonge, DP, Pashley, CH and Gant, TW (2014) Amplicon -based Metagenomic Analysis of Mixed Fungal Samples using Proton Release Amplicon Sequencing. PLoS One. ISSN 1932-6203

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Next generation sequencing technology has revolutionised microbiology by allowing concurrent analysis of whole microbial communities. Here we developed and verified similar methods for the analysis of fungal communities using a proton release sequencing platform with the ability to sequence reads of up to 400 bp in length at significant depth. This read length permits the sequencing of amplicons from commonly used fungal identification regions and thereby taxonomic classification. Using the 400 bp sequencing capability, we have sequenced amplicons from the ITS1, ITS2 and LSU fungal regions to a depth of approximately 700,000 raw reads per sample. Representative operational taxonomic units (OTUs) were chosen by the USEARCH algorithm, and identified taxonomically through nucleotide blast (BLASTn). Combination of this sequencing technology with the bioinformatics pipeline allowed species recognition in two controlled fungal spore populations containing members of known identity and concentration. Each species included within the two controlled populations was found to correspond to a representative OTU, and these OTUs were found to be highly accurate representations of true biological sequences. However, the absolute number of reads attributed to each OTU differed among species. The majority of species were represented by an OTU derived from all three genomic regions although in some cases, species were only represented in two of the regions due to the absence of conserved primer binding sites or due to sequence composition. It is apparent from our data that proton release sequencing technologies can deliver a qualitative assessment of the fungal members comprising a sample. The fact that some fungi cannot be amplified by specific ‘‘conserved’’ primer pairs confirms our recommendation that a multi-region approach be taken for other ampliconbased metagenomic studies.

Item Type: Article
Additional Information: © 2014 Tonge et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Uncontrolled Keywords: fungal spores; fungi; metagenomics; polymerase chain reaction; aspergillus fumigatus; fungal genomics; aspergillus flavus; fungal genetics
Subjects: Q Science > QH Natural history
Divisions: Faculty of Natural Sciences > School of Life Sciences
Depositing User: Symplectic
Date Deposited: 08 Apr 2019 14:06
Last Modified: 15 Apr 2019 15:21

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