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The Use of Next Generation Sequencing to Study the Environmental Mycobiome and Its Potential Health Effects

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The Use of Next Generation Sequencing to Study the Environmental Mycobiome and Its Potential Health Effects The use of next generation sequencing to study the environmental mycobiome and its potential health effects Emma Marczylo Bioaerosols – June 2017 Overview • Brief Background: • Why are CRCE interested in fungal bioaerosols? • Mycobiome analysis: • Why use next generation sequencing? • Ongoing work • What is the current focus of CRCE’s bioaerosol research? 2 Bioaerosols – June 2017 Why bioaerosols? • Respiratory health/toxicology a big focus within our department & bioaerosols represent a current respiratory health concern 3 Bioaerosols – June 2017 Why bioaerosols? • Common sources relevant to public health 4 Bioaerosols – June 2017 Why bioaerosols? • Public concern over health effects of living near composting and intensive farming sites • Systematic reviews on exposures and health outcomes related to bioaerosol emissions from composting facilities (published 2015*) or intensive farming (ongoing) in collaboration with SAHSU • Evidence on both exposure assessment and health effects are limited • A big unknown is the microbial composition of such bioaerosols *Pearson et al, 2015, J Toxicol Environ Health B Crit Rev,18:43-69 5 Bioaerosols – June 2017 Why fungi? Normally die rapidly due to water evaporation, although increased humidity and clumping can prolong survival • Can remain viable for much longer periods, even at low humidity & high/low temperatures • Much less known about the fungal composition of bioaerosols (and other samples) • Fungi linked with development and exacerbation of asthma symptoms 6 Bioaerosols – June 2017 Mycobiome analysis • Mycobiome = fungal composition (eg air, soil, oral swab) • Traditional methods for identifying fungi rely on culture or morphological identification by microscopy • Culture-dependant methods are biased towards fungi that can be cultured (around 17% of known fungi) • Microscopy can be difficult as many fungi cannot be distinguished from each other based upon morphology alone 7 Bioaerosols – June 2017 Mycobiome: NGS method Fungi/spores DNA: ribosomal RNA genes 5.8S Extract DNA PowerSoil® & PureLink™ DNA isolation kits Phusion™ P Ion high-fidelity C R Xpress™ DNA Sample 1 Sample 2 barcodes polymerase BC1 BC4 Aspergillus Sample 1 fumigatus Ion Torrent™ BC2 BC4 BC3 Ion S5 ™ BC2 BC1 BC1 BC5 BC1 Epicoccum BC1 BC2 BC2 BC2 BC4 nigrum BC3 BC3 BC2 BC5 Penicillium BC1 BC6 Sample 2 chrysogenum BC6 NGS + Bioinformatics BC5 BC4 BC3 BC6 BC4 USEARCH BC4 BC3 BC5 Alternaria BC5 BC5 BC6 BC3 BC6 alternata BC6 Database of sample mycobiomes Barcoded PCR products 8 Bioaerosols – June 2017 Mycobiome: Environmental & human samples Soil samples: Indoor: Human: • Woodland • Compost bin (in • Oral/dental (me) kitchen) • Bathroom • Scrubland near (tiles/bath) river 9 Bioaerosols – June 2017 Mycobiome: Environmental & human samples 10 Bioaerosols – June 2017 Current bioaerosol research focus 11 Bioaerosols – June 2017 Acknowledgements PHE SAHSU • Tim Gant • Anna Hansell • Sarah Robertson • Pippa Douglas • Gillian Smith • Alex Elliot University of Adelaide • Giovanni Leonardi • Martin Breed Keele University • Daniel Tonge THANKS! 12 Bioaerosols – June 2017 .
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