IM-BOL Global house dust survey• What is the indoor fungal diversity on a world scale?• What is the correlation in the profile of species detected bydifferent methods?• Are we talking about unculturable or uncultured organisms?• What is the biological significance of the species detected?
Next generation DNA sequencing • Millions of sequences directly from a sample – Culture independent • In fungi, usually ITS +/- 28S • Many platforms and technologies – 454 pyrosequencing – Illumina sequencing – Ion Torrent 18S 5.8S 5.8S 8 25- 25-28SIGS ITS1 S ITS1 ITS2 ITS2 S IGS
Global dust survey: Pyrosequencing results ITS (1 and 2) LSU • 7,032 OTUs Sequence Length 397 bp 445 bp • 56% represented once Mean Total Reads 288,361 282,883 (204,000) (183,000)Amend et al., 2010. Proc. Nat. Acad. Sci. 107, 13748-13753.
Hoekstra et al. 1994. In Health implications of fungi inindoor environments. ed. Samson et al. pp. 169-177.
Wallemiomycetes Sordariomycetes LeotiomycetesDothideomycetes Eurotiomycetes Tremellomycetes Classical isolation study - Netherlands454 pyrosequencing study – Northern hemisphere dy Nort y ? Data reformatted from: Hoekstra et al. 1994. In Health implications of fungi in indoor environments. ed. Samson et al. pp. 169-177. Amend et al., 2010. Proc. Nat. Acad. Sci. 107, 13748- 13753.
454 sequences … Cladosporium Alignment minus 170 bases Reference alignment from TreeBase: Schubert et al. 2009. Persoonia, 22: 111-122.Full alignment
454 sequences… AlternariaReference alignment from: Pryor, B. M., and Gilbertson, R. L.2000. Mycological Research 104: 1312-1321.
454 sequences … Epicoccum In house ITS data plus GenBank data
… a problem with unidentified sequencesFX2FH6V03C9FN4 – ID = DikaryaATCCCTACCTGATCCGAGGTCAACCGTAGAAATGGGGGTTTCTGGAGGCGGGCGGCGCCGAACCTGGAAAGCTGGAGAATTTACTACGCTTGAGGTTCAACACCACCGCCGAGGCCTTTAGGGAGCGTCCGCGACAGGGACGGCACCCAATACCAAGCAGGGCTTGAAGGTTGATAATGACGCTCGAACAGGCATGCTCCCCGGAATACCAGGGAGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAG
Friday Afternoon Mycologist / Kathie Hodge / Kent Loeffler Cornell Mushroom Blog
Keith’s house. Pyro-fungi. Conifer endophytes600 unique sequences53 unassigned to taxonomic order (<10%)
www.cbs.knaw.nl/indoor • now includes classical isolations only • d2e isolations to be added • to be linked with MoBEDAC
IM-Bol conclusions• Vast increase in indoor fungal species diversity on the global scale• Enhanced importance of the class Dothideomycetes – especially Epicoccum, Phoma and Alternaria• Confirmed importance of the class Eurotiomycetes – especially Penicillium and Aspergillus• Confirmed significance of the genus Wallemia – undoubtedly several species rather than one
Concluding thoughts• Identifications in environmental DNA studies – Very sensitive to data gaps and taxonomic imprecision – Especially Last Common Ancestor analysis – Results can vary day by day!• Dilution to extinction – Very labour intensive – Isolates common as well as rare, unusual fungi – Including species not detected by pyrosequencing• Determining biological significance requires multifaceted approach – Living growing moulds vs transients or dead cells – Are all fungi really detected by 454 sequencing?
microbial culturing (conventional & d2e)field biology DNA detection (in a house) (pyrosequencing) comprehensive DNA barcode databases
pyrosequencing direct observation alternative culturing culturing
Thanks to IM-BOL: The Indoor Alfred P. Sloan Foundation of Life Mycota Barcode Real moulds and virtual moulds in your house Genome Canada, NSERC, Canadian Network for DNA Barcoding, Consortium for the Barcode of Life Keith A. Seifert, Joey Tanney, Kalima Mwange, Hai Nguyen, Ed Whitfield Biodiversity, Agriculture & Agri-Food Canada, Ottawa, Canada ECORC colleagues: Tom Graefenhan, Wen Chen, Chris Lewis, Robert A. Samson, Martin Meijer, Vincent Robert André Lévesque CBS Fungal Biodiversity Centre, Utrecht, the Netherlands Thomas D. Bruns, Anthony Amend Plant & Microbial Biology, University of California at Berkeley, USAKeith Seifert email@example.com