1. MBTA (Boston) City Sampling Day 2016 Erica Marie Hartmann Civil and Environmental Engineering Northwestern University Biology and the Built Environment Center University of Oregon Harvard T. H. Chan School of Public Health The Broad Institute

2. Boston: City Sampling Day Goals: Determine if commonly touched surfaces harbor live microbes. If so: Determine which surfaces carry more live/dead microbes. Understand how these microbes survive, and for how long. Generate a PMA-based protocol to determine live/dead microbes compatible with sequencing and swabs

3. Boston Subway Projects: A History Boston subway was swabbed in May and October 2013 Swabbing Protocol: Cotton swabs from Puritan Swabbing solution: 0.15 NaCl with 0.1% Tween 20 Dip cotton swabs in solution, swab 15s, sample each surface 2-3 times Pool swabs for extraction Goals: Profile trains and touchscreens First Boston Subway Study: MetaSUB CSD16: Boston subway was swabbed on June 21, 2016 Swabbing Protocol: Copan and Eswabs from Copan Swabbing solution: For flocked swabs, 0.15 NaCl, for Eswabs, liquid amies Dip cotton swabs in solution, swab 15s, sample each surface 2-3 times Pool swabs for extraction Goals: Characterize multiple surfaces for live/dead taxa

4. TimeSchedule 9:00 amGo to lab and set up negative control (empty cup) and start cultures for PMA controls. 11:00 amMeet sampling team + MBTA staff at Alewife. 11:40 amFinish swabbing touchscreens. Regroup and board Red Line. Deployed first air filter. 12:00 pmSwab Red Line seats and grips. 12:20 pmGet off Red Line at Park Street and go to swab touchscreens. Close first air filter. 12:35 pmFinish at Park Street, get on Green Line toward Lechmere. Deploy second air filter. 12:50 pmGet to Lechmere and unlock empty train. 1:30 pmFinish at Lechmere, ride train back to Brigham Circle. Close second air filter. 2:00 pmGet back to HSPH, store all materials in 4C refrigerator. 4:00 pmBegin PMA treatment. 9:30 pmFinish PMA treatment, store in -20C. Do last negative control and positive control.

5. Study Design Minimize swabbing time in order to prevent too many microbes “dying along the trip” Is this a real problem though? Sample multiple surfaces Trains: Chose grips, seats, and walls (based on previous DNA yield) Stations: Touchscreens and sides of machines Problems PMA is a low-throughput assay Each sample must be done in duplicate Collected 128 swabs with 4 people 64 Eswabs, 64 Copan flocked swabs Swabbed touchscreens, sides of machines, seats, grips, and walls For each set of swabs we have: 4 touchscreens and 4 machine sides at Alewife 4 chairs, 4 grips, and 2 walls on Red Line 2 touchscreens and 2 machine sides at Park 4 chairs, 4 grips, and 2 walls on Green Line Half will be treated with PMA 2 swabs per sample Split into 2 teams, 1 for each set of swabs

6. Alewife: Swabbing touchscreens Decided beforehand the surfaces we wanted to swab For stations, we only swabbed touchscreens (TS) and sides of the machines (TS-W) Handed out “place maps” to swabbers Good place to start to “get used” to the app and swabbing

7. Park Street: Where black swabs mean brake dust Behind the barriers to the left are more machines. Park Street was quite distinct from the rest of the MBTA samples for the original paper The swabs were very dirty, but the MBTA Instructor told us it was brake dust

8. The “Green Line” Originally, the plan was to ride the Green Line from Park Street down to Brigham Circle Would give us ~20 minutes to collect 20 samples MBTA Instructor suggested he unlock some Green Line trains that had been used for rush hour sitting at Lechmere. We would have much more time. He unlocked two cars. The first car didn’t match the map I had originally drawn, the second one did. First Car Second Car

9. Lessons Learned from CSD16 GIS Cloud Crashed after photo taking for the Galaxy S4, fine on the iPhone Autofill or ability to cut/paste would be useful Battery drain Trains MBTA staff can possibly allow us onto rush hour trains taken out of service depending on time of swabbing. Should take more time to take photos, but train runs can be very hectic and difficult to complete (rushing before stop, and many people to talk to) PMA Assays Takes incredibly long: 5-6 hours to handle 128 swabs (treated half with PMA, half without) Time-consuming steps: Cutting the swabs to fit in an Eppendorf tube (or we need to find a centrifuge that fits the swab tubes) Only 1 centrifuge and set of lights, no multichannel pipets, can only handle 24 at a time. Questions: Should the liquid amies be isolated as well? Should controls be subjected to “PMA” negative treatment? It does lower DNA yield. Need official assay if Eswabs are to be used. Made one up the day of.

10. Thanks! Tiffany Hsu thsu@fas.harvard.edu Curtis Huttenhower chuttenh@hsph.harvard.edu

11. 1.Start at Park Street and collect touchscreen samples. 2.Take E line to Brigham Circle. 3.Collect train samples en-route. 1.Include surfaces for seat, seat back, grips, horizontal/vertical poles, and wall. 4.Take samples to lab for isolation and PMA treatment.

12. On sampling: Each sample will require 4 swabs Samples need to be collected twice, once with PMA (propidium monoazide) and once without To ensure enough DNA, two swabs are required for each sample type Potential Issues: Sampling collection time: If more time is needed to collect samples, collect samples on the way to Park Street and on the way back Protocol limitations: Determine if PMA-treated samples can be safely stored in the -20C before DNA isolation. If not, will have to sample over multiple days. Decide if samples should be treated with PMA during collection or after.

13. Riding to Park Street: Swabbing trains Train was relatively empty at Alewife, and got more and more crowded as we approached Park Street. Already knew only had ~15 minutes for each team to collect 20 swabs Fewer photos and documentation because of this Unfortunately, got on a train with leather grips