Iden%fying pro%sts in NYC subway samples Julia Maritz Carlton Lab New York University Center for Genomics and Systems Biology An NYU Grand Challenge Project: Mapping the New York City “MetaGenome” coronavirus Lactobacillus Bacillus cereus Methanosarcina Cryptosporidium Stachybotrys Pro?sts are found through out the eukaryo?c tree of life Focusing on one parasi%c disease today: • Single-celled eukaryotes malaria • Free-living environmental pro%sts Blastocystis • Human/animal commensals and Toxoplasma parasites SAR Cryptosporidium Rhizaria Stramenopiles Cercozoa Radiolaria Foraminifera Alveolates Centrohelid Glaucophytes Red algae Haptophytes Green algae & land plants Cryptophytes Entamoeba Apusoza Breviata Discoba Oxymonads Trimasx Malawimonas Excavata Diplomonads Retortamonads Parabasalids Collodictyonids Eukaryotes Giardia Prokaryotes Trichomonas Bacteria Archaea (aer Roger & Simpson, 2009. Current Biology 19: R165-167.) Many pro?sts are zoono?c • Transmissible between humans, domes%c animals and wildlife • 16-20% of emerging zoonoses • Many are diges%ve and reproduc%ve parasites • Cause global poor health and economic losses Blastocystis Giardia intestinalis Cryptosporidium Entamoeba histolytica Trichomonads Toxoplasma gondii Tritrichomonas foetus • Despite this, emerging parasi%c zoonoses are neglected diseases Animal Disease Incidence Consequences • Few studies have inves%gated their emergence and spread in Cats Diahrrea ~1/3 of cats worldwide Non-fatal Cale Vaginosis, Unknown but Spontaneous abor%on, urban environments endometri%s worldwide infer%lity, herd loss Pigs Commensal 68-98% domesc pigs NA Humans Pulmonary infec%ons Unknown Exacerbate symptoms, prolong illness Project goals • Inves%gate populaon dynamics and gene%c diversity of zoono%c pro%sts present in New York City – Use NGS methods to iden%fy, track, and characterize zoono%c pro%st communi%es in NYC – Associate their presence with local wildlife and domes%c animal populaons. Blastocystis Giardia intestinalis Cryptosporidium Entamoeba histolytica Trichomonads Toxoplasma gondii How to idenfy prosts in environmental samples? • 18S rRNA amplicon sequencing – Small subunit of rRNA – Present in all eukaryotes – Has 9 variable regions • V9 and V4 are good candidates – Illumina sequencing technology – V9 • Vertebrate blocking primer • Three domain primers – V4 • Largest variable region • Eukaryote specific Ueda-Nishimura and Mikata 2000 18S protocol tesng and data analysis • DNA from four subway swabs Borough $Microbial$analyses$Collecon site – July 2013 Brooklyn Bedford Ave Turnsle Brooklyn Bedford Ave wooden bench DNA$extracOon$ th • Amplicon PCR Manhaan 8 Ave wooden bench Manhaan 6th Ave turns%le – V9 and V4 regions • 18S paired-end sequencing – Illumina Miseq 16S$/$18S$ribosomal$~200bp ~500bp • Compare results RNA$$ – QIIME based pipeline – de novo OTUs at 98% iden%ty – Curated SILVA reference database community$ structure$ Majority of eukaryotes iden?fied were fungi • 9 major clades, 33 genera Unassigned • Dominated by fungi SAR;__Stramenopiles – 50-90% of each sample SAR;__Rhizaria SAR;__Alveolata • Less metazoan DNA than Opisthokonta;__Metazoa might expected Opisthokonta;__Fungi – Mostly insects and mice Excavata;__Metamonada • Manhaan samples have Excavata;__Discoba higher diversity ArchaeplasFda;__ChloroplasFda Amoebozoa;__Conosa • Numerous pro%sts detected as well Brooklyn ManhaVan Several parasic prosts are present • Zoono%c taxa of interest – BlastocysFs • Gut parasite Unassigned – Toxoplasma gondii SAR;__Stramenopiles • Toxoplasmosis – Trichomonas vaginalis SAR;__Rhizaria • Most common human non-viral STD SAR;__Alveolata – Tritrichomonas foetus Excavata;__Metamonada • Parasite of cats & dogs & humans Excavata;__Discoba • Human/animal commensals Amoebozoa;__Conosa – Entamoeba dispar • Commensal gut taxa • Many unassigned taxa Brooklyn ManhaVan Summary and future work • Pro%sts can be iden%fied in environmental samples with the 18S rRNA gene – Diverse communies – Including many taxa of interest • Lots of uniden%fied sequences – Need be^er databases for iden%ficaon • Collect and sequence other NYC environments: ATMS$in$NYC$ • Sewage • Soil • ATMs Acknowledgements Advisor: Visionaire Staff: DEP Staff: Jane Carlton Dennis Keefe Peter Williamsen Ed Quinones Abeba Negatu Carlton Lab: Michael Gubbins William Kelley Susan Joseph Zach Gallager Trikam Patel Steven Sullivan Manu Patel Theresa Ten Eyck Patrick Jagessar Commiee: Diane Hammerman Genomics Core: Elodie Ghedin Tara Moran Patrick Eichenberger Theresa Ten Eyck Kae Schneider Christopher Mason University of the Pacific: Kirkwood Land .