Amplicon –Based Metagenomic Analysis of Mixed Fungal Samples Using Proton Release Amplicon Sequencing

Gant TW (2014) Amplicon -Based Metagenomic Analysis of Mixed Fungal Samples Using Proton Release Amplicon Sequencing. PLoS ONE 9(4): e93849. doi:10.1371/journal.pone.0093849 Amplicon -Based Metagenomic Analysis of Mixed Fungal Samples Using Proton Release Amplicon Sequencing Daniel P. Tonge 0 Catherine H. Pashley 0 Timothy W. Gant 0 Vishnu Chaturvedi, California Department of Public Health, United States of America 0 1 Centre for Radiation, Chemical and Environmental Hazards, Public Health England , Harwell Campus, Didcot, Oxfordshire , United Kingdom , 2 Department of Infection , Immunity and Inflammation , Institute for Lung Health, University of Leicester , Leicester , United Kingdom , 3 Faculty of Computing, Engineering and Sciences, Staffordshire University , Stoke-on-Trent , United Kingdom 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. - Funding: DPT and TWG are supported by Centre for Radiation, Chemical and Environmental Hazards, Public Health England (Development Grant 108539). CHP is supported by the Midlands Asthma and Allergy Research Association (MAARA) and the National Institute for Health Research Leicester Respiratory Biomedical Research Unit. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The fungal kingdom is highly diverse and includes taxa from numerous different ecological niches with varied life history strategies and morphologies, ranging from unicellular yeasts and microscopic moulds to large mushrooms. The number of formally described fungal species is over 74,000 with greater than 12,000 named species in the British Isles alone, however, estimates of the number of species globally is believed to be at least 1.5 million, but probably as many as 3 million [1,2]. Spores are one of the mechanisms used by fungi for dispersal, most of which are windborne and vary greatly in size, morphology and method of release; with both passive and active spore liberation methods. Fungi are an essential component of the ecosystem and have major economic value, both positively (commercial exploitation for drugs, food-stuff, fuel, pesticides etc. [3]) and negatively as plant and animal pathogens, and play an important role in human health as allergens, pathogens and commensals. Traditional methods for identifying microscopic fungi and airborne fungal spores rely on culture or morphological identification by microscopy. Culture-dependant methods inevitably underestimate diversity, and grossly bias studies towards fungi that can be cultured on generic fungal growth media. Estimates suggest that of the known fungi, only 17% can be readily grown in culture [3] and of those many will produce only sterile mycelium (Bridge and Spooner, 2001). In clinical studies, reliance on culture can miss potentially important fungi [4]. Microscopy is less prone to bias; however, many spores cannot be distinguished from each other based upon their morphology alone. Of the 40 or so fungal categories that can be recognised, some can be classified to genus, a few to species, but many have to be recorded in groups with similar characteristics [5]. The development of molecular techniques, that utilise polymerase chain reaction (PCR) to amplify regions of the fungal genome thought to be specific to a particular species, provided the first culture-independent means of detecting microorganisms. Amplicon-based fungal metagenomic studies target various regions of the nuclear ribosomal operon (rDNA); which is present in multiple copies, contains both highly conserved and variable regions, and for which numerous universal primers exist. The fungal ribosomal operon consists of the small subunit (SSU or 16S/18S), and the large subunit (LSU or 23S/25S/28S), separated by an internal transcribed spacer (ITS) region (comprising two sections, ITS1 and ITS2) that bracket the conserved 5.8S. Three prime of the LSU is the 5S ribosomal gene flanked by two intergenic spacer regions (IGS1 and 2 respectively) [6]. Early culture-free molecular studies of fungi [79] required the amplification of fungal DNA using conserved oligonucleotide primers, and utilised the sequencing method of Sanger to determine the sequence of the resulting amplicons [10]. Sanger sequencing provided the long read length (,1000 bp) and basecalling accuracy required for such analyses, however was unsuitable for the generation of sufficient sequencing data (termed sequencing depth) to resolve complex microbial systems commonly associated with environmental sampling, in a reasonable timeframe. The introduction of next generation sequencing revolutionised microbial metagenomics by providing much greater sequencing depth. This however tended to be at the expense of read length, with some next generation platforms generating sub100 bp reads. The early fungal amplicon-based metagenomic studies relied on expensive 454 sequencing to enable the g (...truncated)