Idenfying prosts 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 Prosts are found through out the eukaryoc tree of life Focusing on one parasic disease today: • Single-celled eukaryotes malaria • Free-living environmental prosts 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 prosts are zoonoc • Transmissible between humans, domesc animals and wildlife • 16-20% of emerging zoonoses • Many are digesve and reproducve parasites • Cause global poor health and economic losses

Blastocystis Giardia intestinalis Cryptosporidium Entamoeba histolytica Trichomonads Toxoplasma gondii

Tritrichomonas foetus

• Despite this, emerging parasic zoonoses are neglected diseases Animal Disease Incidence Consequences • Few studies have invesgated their emergence and spread in Cats Diahrrea ~1/3 of cats worldwide Non-fatal Cale Vaginosis, Unknown but Spontaneous aboron, urban environments endometris worldwide inferlity, herd loss Pigs Commensal 68-98% domesc pigs NA

Humans Pulmonary infecons Unknown Exacerbate symptoms, prolong illness Project goals

• Invesgate populaon dynamics and genec diversity of zoonoc prosts present in New York City – Use NGS methods to idenfy, track, and characterize zoonoc prost communies in NYC – Associate their presence with local wildlife and domesc 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 turnsle – 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% identy – Curated SILVA reference database community$ structure$ Majority of eukaryotes idenfied 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 Archaeplasda;__Chloroplasda

Amoebozoa;__Conosa • Numerous prosts detected as well Brooklyn Manhaan Several parasic prosts are present

• Zoonoc taxa of interest – Blastocyss • 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 Manhaan Summary and future work

• Prosts can be idenfied in environmental samples with the 18S rRNA gene – Diverse communies – Including many taxa of interest

• Lots of unidenfied sequences – Need beer databases for idenficaon

• 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