Transcript City Center
MetaSUB Summit 2016 Shanghai, China July 1-3, 2016 MetaSUB Shanghai Updates 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 [email protected] City Sampling Day (CSD) Shanghai Accomplishments • • • • • • • • • • • 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 -80oC (<24 hrs post collection) 376 stations 14 lines Maglev >600 km 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 朱嗣博 唐安怡 陈炜 陈心影 蔺帆 王晓林 温卓 杨竞成 李斌 李冰樱 张蕾 庆涛 商骏 包玎 侯湾湾 任路瑶 Number of Samples 9.569 million trips per day Time 2 Google Map Wetland XJW Station x 4 Town Center City Center WJC Station x 4 RMGC Station x 4 Pilot Study: 12 Samples 3 stations 4 replicates per station March 17 & 27, 2016 3 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 Sampling DNA Extraction • MoBio DNA Extraction Kit • Library Preparation • Nextera XT 96 Kit Library Preparation • Sequencing • Illumina X10 Sequencer • 150 PE • 60 M reads/sample DNA Sequencing • Data Mining • Diamond Blasting • Megan LCA Analysis Data Mining 4 DNA Extraction: Yield/Swab Is Station Dependent DNA Yield (ng/swab) 18 City Center 16 14 12 Town Center 10 Wetland (New, less traffic) 8 6 4 2 0 R1 R2 R3 R4 X1 X2 X3 X4 W1 W2 W3 W4 Swab Number MS00101 MS00102 MS00103 MS00104 MS00105 MS00106 MS00107 MS00108 MS00109 MS00110 MS00111 MS00112 Collector Site TAY TAY TAY TAY ZSB ZSB ZSB ZSB CW CW CW CW RMGC RMGC RMGC RMGC XJWC XJWC XJWC XJWC WJC WJC WJC WJC Seq Name R1 R2 R3 R4 X1 X2 X3 X4 W1 W2 W3 W4 DNA Amount (ng/swab) 11.5 16.25 9 17 1.74 1.81 1.99 2.07 6.95 3.73 8.2 5.55 DNA yield per swab was much higher in RMGC station (City Center), the busiest metro station in Shanghai. 5 Quality of Libraries 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. 6 Quality of Sequencing Data • Good Q30 per base (>90%) Read1 of RMGC1 GC Content of RMGC1 R1 • Good Read1/2 Q30 score • Normal GC distribution • 60 million reads per sample Read2 of RMGC1 GC Content of RMGC1 R2 7 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. Raw counts at species, genus, and class levels were normalized according to: A= Log2(CPM+1) 8 PCA of 12 Samples Class Genus Species 9 Number of Species, Genera and Classes Detected across 12 Samples Class Genus Species Metagenome composition (Biodiversity): City Center < Town Center < Wetland 10 Overlap of Top 100 Genera RMGC XJWC WJC Between two biological replicates: ~70% Between two stations: ~50% 11 Bacteria Dominant Metagenome RMGC City Center WJC Town Center XJWC Wetland • Bacteria were dominant across all samples. 12 Heatmap at Kingdom Level Viridiplantae Vitis Homo Bos Cricetulus Amoeba Pleosporineae Leotiomyceta (Penicillin) Malassezia Lentivirus Hepadnaviridae (HBV) Bacteria Phage and Herpes Virus RMGC WJC XJWC 13 Take Home Messages • 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. 14 Acknowledgments 1. 朱嗣博 2. 唐安怡 3. 陈炜 4. 陈心影 Anyi TANG 5. 蔺帆 Tao QING 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 Sibo ZHU Wanwan HOU 16.任路瑶 15