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 Trimas x
Malawimonas Excavata Diplomonads Retortamonads
Parabasalids Collodictyonids Eukaryotes Giardia Prokaryotes Trichomonas Bacteria Archaea (a er 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 Ca le Vaginosis, Unknown but Spontaneous abor on, urban environments endometri s worldwide infer lity, herd loss Pigs Commensal 68-98% domes c pigs NA
Humans Pulmonary infec ons Unknown Exacerbate symptoms, prolong illness Project goals
• Inves gate popula on 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 popula ons.
Blastocystis Giardia intestinalis Cryptosporidium Entamoeba histolytica Trichomonads Toxoplasma gondii How to iden fy pro sts 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 tes ng and data analysis
• DNA from four subway swabs Borough $Microbial$analyses$Collec on site – July 2013 Brooklyn Bedford Ave Turns le Brooklyn Bedford Ave wooden bench
DNA$extracOon$ th • Amplicon PCR Manha an 8 Ave wooden bench Manha an 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
• Manha an samples have Excavata;__Discoba
higher diversity Archaeplas da;__Chloroplas da
Amoebozoa;__Conosa • Numerous pro sts detected as well Brooklyn Manha an Several parasi c pro sts are present
• Zoono c taxa of interest – Blastocys s • 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 Manha an Summary and future work
• Pro sts can be iden fied in environmental samples with the 18S rRNA gene – Diverse communi es – Including many taxa of interest
• Lots of uniden fied sequences – Need be er databases for iden fica on
• 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 Commi ee: Diane Hammerman Genomics Core: Elodie Ghedin Tara Moran Patrick Eichenberger Theresa Ten Eyck Ka e Schneider Christopher Mason University of the Pacific: Kirkwood Land