1. MetaSUB Shanghai Updates MetaSUB Summit 2016 Shanghai, China July 1-3, 2016 Sibo Zhu, Anyi Tang, Tao Qing, Wanwan Hou, Lei Zhang, Wei Chen, Ding Bao, Xiaolin Wang, Yuanting Zheng, and Leming Shi Center for Pharmacogenomics Fudan-Zhangjiang Center for Clinical Genomics Shanghai Big Data Center for Precision Medicine School of Life Sciences, Fudan University Shanghai, China +86-186-1682-7008 LemingShi@Fudan.edu.cn

2. City Sampling Day (CSD) Shanghai Accomplishments 2 748 samples were collected 374/376 (99.47%) stations sampled 2 replicates per station Site: Elevator handrails Date: June 21, 2016 Time: 9:00 to 19:00 -80 o C (<24 hrs post collection) Number of Samples Time 376 stations 14 lines Maglev >600 km 1. 朱嗣博 2. 唐安怡 3. 陈炜 4. 陈心影 5. 蔺帆 6. 王晓林 7. 温卓 8. 杨竞成 9. 李斌 10. 李冰樱 11. 张蕾 12. 庆涛 13. 商骏 14. 包玎 15. 侯湾湾 16. 任路瑶 9.569 million trips per day

3. X JW Station x 4 W JC Station x 4 R MGC Station x 4 Google Map City Center Wetland Town Center 3 Pilot Study: 12 Samples 3 stations 4 replicates per station March 17 & 27, 2016

4. Shanghai Pilot Study with 12 Samples 3 Stations in Shanghai Renminguangchang (RMGC), Wujiaochang (WJC), and Xinjiangwancheng (XJWC) 4 biological replicates per station Sample Collection COPAN C480 swab Handrail of elevators March 17 and 27, 2016, 6:00~7:00 p.m. DNA Extraction MoBio DNA Extraction Kit Library Preparation Nextera XT 96 Kit Sequencing Illumina X10 Sequencer 150 PE 60 M reads/sample Data Mining Diamond Blasting Megan LCA Analysis Sampling 4 DNA Extraction Library Preparation DNA Sequencing Data Mining

5. DNA Extraction: Yield/Swab Is Station Dependent 5 Swab Number CollectorSite Seq Name DNA Amount (ng/swab) MS00101TAYRMGCR1 11.5 MS00102TAYRMGCR2 16.25 MS00103TAYRMGCR3 9 MS00104TAYRMGCR4 17 MS00105ZSBXJWCX1 1.74 MS00106ZSBXJWCX2 1.81 MS00107ZSBXJWCX3 1.99 MS00108ZSBXJWCX4 2.07 MS00109CWWJCW1 6.95 MS00110CWWJCW2 3.73 MS00111CWWJCW3 8.2 MS00112CWWJCW4 5.55 DNA yield per swab was much higher in RMGC station (City Center), the busiest metro station in Shanghai. City Center Town Center Wetland (New, less traffic)

6. Quality of Libraries 6 Two libraries from RMGC samples were taken as examples, with a DNA peak ranging from 300-1,000 bp showing a successful fragmentation with Tn5.

7. Quality of Sequencing Data Read1 of RMGC1 Read2 of RMGC1 GC Content of RMGC1 R1 GC Content of RMGC1 R2 Good Q30 per base (>90%) Good Read1/2 Q30 score Normal GC distribution 60 million reads per sample 7

8. Data Analysis Pipeline Low-quality reads and sequencing adapters were filtered with Trimmomatic (version 0.33). High quality reads were aligned to the NCBI nr database using a sequence comparison program Diamond. The resulting data were processed with MEGAN6 to produce an interactive analysis of over 660,000 taxa, including approximately 25,000 prokaryotes, 84,000 animals, 65,000 plants, and 17,000 viruses. The individual species were hierarchically grouped into clades at the levels of: Superkingdom, Kingdom, Phylum, Class, Order, Family, Genus, and Species. 8 Raw counts at species, genus, and class levels were normalized according to: A= Log 2 (CPM+1)

9. PCA of 12 Samples 9 ClassSpeciesGenus

10. Number of Species, Genera and Classes Detected across 12 Samples Metagenome composition (Biodiversity): City Center < Town Center < Wetland 10 ClassSpeciesGenus

11. Overlap of Top 100 Genera 11 RMGCWJCXJWC Between two biological replicates: ~70% Between two stations: ~50%

12. Bacteria Dominant Metagenome RMGCWJCXJWC Bacteria were dominant across all samples. 12 City Center Town Center Wetland

13. Bacteria Phage and Herpes Virus Amoeba Viridiplantae Pleosporineae Leotiomyceta (Penicillin) Hepadnaviridae (HBV) Lentivirus Malassezia HomoBos Cricetulus Vitis 13 Heatmap at Kingdom Level RMGCWJCXJWC

14. 748 samples were collected according to standard protocol on CSD. In the pilot study, three stations each with 4 replicates were studied. Bacteria were dominant across samples. The metagenome from XJWC station was more biodiverse at Class, Genus and Species levels compared to other stations, with more plants and fungi detected. This observation appears to be consistent with the fact that the XJWC station was newly built at a wetland. Take Home Messages 14

15. Acknowledgments 15 Sibo ZHUWanwan HOU Anyi TANG Tao QING 1. 朱嗣博 2. 唐安怡 3. 陈炜 4. 陈心影 5. 蔺帆 6. 王晓林 7. 温卓 8. 杨竞成 9. 李斌 10. 李冰樱 11. 张蕾 12. 庆涛 13. 商骏 14. 包玎 15. 侯湾湾 16. 任路瑶