"The prospects for Nextgen surveillance of pathogens: A view from a Public Health Lab" presentation at the Standards for Pathogen Identification via NGS (SPIN) workshop hosted by the National Institute for Standards and Technology in October 2014 by William Wolfgang, PhD from Wadsworth Center NYSDOH.

1. The prospects for Nextgen surveillance of
pathogens: A view from a Public Health Lab
William Wolfgang
Wadsworth Center NYSDOH
NIST Workshop 10/20/14

2. Public Health Genomics – projects at the
Wadsworth
Project( Sample(Source( PI(
Salmonella(surveillance+ Isolate+ Wolfgang+
TB(surveillance+and+drug+
resistance+
Isolate+and+
Primary+
Escuyer+and+
Musser+
C.(Botulinum((source+tracking+ Isolate+ Egan+
HCV+subtyping++ Primary+ Parker+and+
Chou+
Adenovirus(surveillance(and(+
characteriza@on+ Isolate+ St.+George+
and+Lamson+
Rabies(virus(intraEhost+evolu@on+ Isolate+ Davis+
Mosquito+microbiome+and++
West+Nile+Virus+ Primary+ Ciota+and+
Kramer+

3. We collaborate with a number of different
groups on Pathogen sequencing projects
• Global Microbial Identifier (GMI) initiative
• CDC: Listeria monocytogenes initiative and AMD initiative
• FDA: GenomeTrakr initiative
• Minnesota and Washington Departments of Health
• And we hope to do more

4. At the Wadsworth we need standards
• As we translate to new technologies we need to know:
• Faster
• Cheaper
• Better
• For this we need to make accurate and meaningful comparisons.
• To do this we need standards.

5. Why use Nextgen for Salmonella typing?
• PFGE has low discriminatory power.
.
Each year
• 1 million cases Salmonella in US.
• 19,000 hospitalizations and 378 deaths.
• The Wadsworth receives about 1,500/yr.

6. Greater discrimination can be achieved by
Whole Genome Sequencing

7. Proof of principle study on a Salmonella
Enteritidis outbreak
• Sept. 2010 Connecticut Dept. of Health identifies a Salmonella
outbreak in a long term care facility (LTCF).
• Outbreak was linked to cannoli from a Westchester bakery.
• Both NY and CT cases consumed cannoli’s.
• Isolates had the most common PFGE pattern.

8. Retrospective cohort
Key County Date PFGE
IDR1000029153 Cattaraugus 8/10/10 JEGX01.0004
IDR1000031528 Rockland 8/26/10 JEGX01.0004
IDR1000033213 Putnam 9/10/10 JEGX01.0004
IDR1000033369 Putnam 9/10/10 JEGX01.0004
IDR1000033371 Putnam 9/11/10 JEGX01.0004
IDR1000034601 Washington 9/13/10 JEGX01.0004
IDR1000034587 Westchester 9/20/10 JEGX01.0004
IDR1000035417 Putnam 9/22/10 JEGX01.0004
IDR1000035178 Westchester 9/13/10 JEGX01.0004
IDR1000035179 Greenwich CT 9/12/10 JEGX01.0004
IDR1000035180 Westchester 9/12/10 JEGX01.0004
IDR1000035181 Westchester 9/13/10 JEGX01.0004
IDR1000035182 Westchester 9/12/10 JEGX01.0004
IDR1000035183 Greenwich CT 9/16/10 JEGX01.0004
IDR1000036119 9/17/10 JEGX01.0004
IDR1100035184 Westchester 9/16/10 JEGX01.0004
IDR1000036319 Putnam 9/28/10 JEGX01.0004
IDR1000036979 Putnam 10/8/10 JEGX01.0004
IDR1000038792 Nassau 10/29/10 JEGX01.0004
IDR1000034599 Orange 9/15/10 JEGX01.0004
IDR1100006235 Westchester 2/21/11 JEGX01.0004
IDR1100021079 Rockland 7/13/11 JEGX01.0004
IDR1000030147 Out-Of-State 8/22/10 JEGX01.0004
IDR1100003844 Onondaga 2/1/11 JEGX01.0004
IDR1100022186 Yates 7/22/11 JEGX01.0004
IDR1100027690 Erie 9/6/11 JEGX01.0004
IDR1100030508 Madison 10/9/11 JEGX01.0004
IDR1100031312 Suffolk 10/5/11 JEGX01.0004
IDR1100032014 Onondaga 10/22/11 JEGX01.0004
IDR1000028670 Nassau 8/8/10 JEGX01.0004
IDR1000029949 Suffolk 8/16/10 JEGX01.0004
IDR1000033603 Erie 9/14/10 JEGX01.0004
IDR1000034213 Erie 9/13/10 JEGX01.0004
IDR1000037723 Westchester 10/4/10 JEGX01.0004
IDR1000039087 Westchester 10/27/10 JEGX01.0004

9. Whole genome Cluster Analysis ( WGCA)
can identify an outbreak cluster not detected
88
7.3 SNPs
11­06235
10­33603
10­35184
11­27690
10­37723
10­35183
10­36319
+
10­31528
10­33369
+
10­35417
+
11­32014
10­33213
+
10­34599
10­34587
+
10­35179
11­22186
11­30508
10­30147
10­34601
+
11­03844
10­34213
10­33371
+
10­35181
10­36119
10­28670
11­21079
10­35178
10­35182
10­29153
11­31312
10­38792
+
10­36979
+
10­29949
10­39087
10­35180
100
100
100
85
88
68
100
100
A
B
LTCF
by PFGE
All isolates are PFGE PATTERN 4

10. Implementing WGCA for SE in real-time.
• Evaluate WGCA compared to PFGE.
• Speed - Faster
• Cost - Cheaper
• More Actionable Clusters – Better
• Develop an in house bioinformatics pipeline.
• Develop communication pipeline to epidemiologists.
• Determine cluster parameters that represent an outbreak from a single
source (assign a probability).
• Use data sets to evaluate evolving informatic methods.
• Become proficient (PT programs).

11. Over the past 12 months
• Sequenced all Salmonella Enteritidis (379 genomes).
• All data at NCBI
• Developed an in House pipeline to analyze the data.
• SNP based phylogenetic trees were constructed in real time.
• 63 phylogenetic clusters were reported to epidemiologists.
• 0 to 5 snps differences

12. Data is
analyzed using
a portal
developed by
Informatics core

13. In House Developed Pipeline

14. Current tree
• Is this tree structure
reproduced by other
pipelines?
• Is it reproduced within
our pipeline?
• What are the minimal
sequencing metrics
required to create
reproducible trees?
• What changes would
make this faster-cheaper-
better.
*
swgs1063
8621sgws-YN
3821sgws-YN
swgs1029
swgs1007
swgs1068
swgs1054 4843100031RDI-YN
NY-SWGS1249
NY-swgs1266
NY-NY-swgs1332
swgs1280 NY-swgs1348
NY-swgs1404 NY-swgs1365
NY-swgs1382
NY-swgs1296
swgs1080 NY-swgs1331
swgs1371 PFGE Type
NY-swgs1275
NY-*=2
*=4
*= 5
NY-swgs1421
*=21 or 23
*=34
*=56
swgs1041 swgs1077 9570100031 RDI YN -NY-swgs1294 swgs1083 swgs1016
swgs1003 swgs1085
swgs1072 NY-swgs1269
swgs1035 NY-swgs1295
NY-swgs1278 NY-swgs1279
0.02
NY-swgs1314
swgs1212
NY-swgs1454
NY-swgs1307
NY-swgs1341
0531sgws-YN 0631sgws-YN
NY-swgs1359
NY-swgs1377
NY-swgs1416
NY-swgs1276
NY-swgs1336
swgs1033
NY-swgs1368
swgs1065
NY-swgs1274
NY-swgs1470
NY-swgs1240
swgs1039
NY-swgs1476
NY-swgs1373
0321sgws
4421sgws-YN
NY-swgs1402 NY-swgs1361
1641sgws-YN
swgs1205
swgs1038
NY-swgs1334
swgs1047
NY-swgs1401
swgs1048
NY-swgs1369
swgs1012
swgs1034
swgs1061
NY-swgs1347
swgs1021
NY-swgs1397
swgs1025
NY-swgs1346
swgs1014
swgs1088
swgs1008
NY-swgs1467
NY-swgs1345
NY-swgs1381
swgs1030
swgs1207
swgs1031
NY-swgs1236
NY-swgs1363
NY-swgs1356
NY-swgs1247
NY-swgs1384
NY-swgs1427
NY-swgs1405
37 38000031RDI-YN
NY-swgs1422
swgs1210
NY-swgs1328 NY-swgs1325
NY-swgs1386
swgs1023
swgs1208
NY-swgs1241
NY-swgs1445
NY-swgs1255
NY-swgs1292
NY-swgs1407 NY-swgs1259
NY-swgs1447
NY-swgs1468
swgs1049
swgs1203
7071100031RDI-YN
swgs1036
NY-swgs1327 NY-swgs1311
swgs1053
NY-swgs1261
swgs1010 NY-swgs1293
swgs1024
NY-swgs1395
swgs1214
swgs1095
NY-swgs1398
NY-swgs1349
NY-swgs1453
swgs1086
NY-swgs1455
NY-swgs1282
swgs1073
swgs1217
NY-swgs1450
swgs1198
swgs1221
NY-swgs1267
NY-swgs1319
NY-swgs1329
swgs1197
swgs1055
swgs1067
NY-swgs1414
6418000031RDI-YN
NY-swgs1239
NY-swgs1357
swgs1194
swgs1046
swgs1060
NY-swgs1390
swgs1200
swgs1087
swgs1216
NY-swgs1310
NY-swgs1410
NY-swgs1352
8731sgws-YN
swgs1066
swgs1064
NY-swgs1342
NY-swgs1370
swgs1222
NY-IDR1300012602 NY-swgs1321
NY-swgs1431
NY-swgs1287
swgs1062
NY-swgs1262
NY-swgs1471
NY-swgs1298
NY-swgs1396
NY-swgs1403
NY-swgs1285
swgs1022
NY-swgs1415 NY-swgs1242
swgs1057
swgs1019
swgs1074
NY-swgs1302r
NY-swgs1312
NY-swgs1408
NY-swgs1433
NY-swgs1385
swgs1092
swgs1050
NY-swgs1358
NY-swgs1246
swgs1233
NY-swgs1308r
NY-swgs1288
NY-SWGS1248
swgs1009R
NY-swgs1412
swgs1229
NY-swgs1243
swgs1042
swgs1005
swgs1078
swgs1094
NY-swgs1374
5968000031RDI-YN
NY-swgs1309
NY-swgs1463
NY-swgs1430
NY-swgs1309r NY-swgs1253
swgs1017
Salmonella_enterica_str_P125109
NY-swgs1335
swgs1051
NY-swgs1472
NY-swgs1281
NY-swgs1460
swgs1213
NY-swgs1426
3431sgws-YN
swgs1020
swgs1059
NY-swgs1337
6631sgws-YN
swgs1199
NY-swgs1256
NY-swgs1400
swgs1081
NY-swgs1300
NY-swgs1303
NY-swgs1409
swgs1028
swgs1076
swgs1084
swgs1204
8241sgws-YN
NY-swgs1429
1001sgws-YN
NY-swgs1399
NY-swgs1324
0221 sgws
swgs1069
swgs1218
NY-swgs1290
5531sgws-YN
swgs1026
swgs1195
NY-swgs1272
swgs1225
swgs1032
swgs1011
swgs1004
NY-swgs1317
NY-swgs1444
NY-swgs1383
783 1sgws-YN 8331sgws-YN
0221100031RDI-YN
NY-swgs1379
NY-swgs1424
NY-swgs1432
NY-swgs1286
NY-swgs1330
swgs1206 swgs1006
swgs1202
NY-swgs1302
NY-swgs1323
NY-swgs1475
6541 sgws-YN
NY-swgs1451
NY-swgs1376
NY-swgs1277
NY-swgs1392
NY-swgs1462
NY-swgs1411
NY-swgs1344
NY-swgs1320
swgs1071
NY-swgs1419
swgs1015
4821sgws-YN
NY-swgs1263
swgs1091
NY-swgs1353
swgs1043
swgs1070
swgs1052
swgs1079
swgs1228
NY-swgs1364
NY-swgs1301
NY-swgs1362
3741 sgws-YN NY-swgs1413
NY-swgs1452
NY-swgs1291
NY-swgs1254
NY-swgs1388
NY-swgs1322
NY-IDR1300011034
NY-swgs1289
NY-swgs1367
NY-swgs1326
3331sgws-YN
swgs1045
644 1sgws-YN
NY-swgs1257
swgs1056
NY-swgs1389
9331sgws-YN
swgs1201
NY-swgs1425
NY-swgs1391 swgs1013
swgs1044
NY-swgs1260
NY-IDR1300009147
swgs1235
NY-swgs1238
swgs1219
swgs1224
swgs1227
NY-swgs1270
NY-swgs1245
NY-swgs1417
swgs1037
NY-swgs1252
swgs1090
swgs1082
NY-swgs1474
swgs1027
NY-swgs1469
NY-swgs1375
NY-swgs1304
NY-swgs1313
swgs1226
3 221sgws
swgs1058
NY-swgs1271
NY-swgs1420
NY-swgs1351
swgs1234 swgs1093
swgs1215
NY-swgs1423 NY-swgs1448
5031sgws-YN
NY-swgs1393
NY-swgs1306
swgs1211
swgs1209
6041sgws-YN
NY-swgs1372
NY-swgs1258
swgs1196
NY-swgs1380
swgs1040
NY-swgs1251
NY-swgs1318
NY-swgs1354
NY-swgs1273
swgs1018
swgs1089
NY-swgs1418
NY-swgs1394
swgs1075
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15. Can we develop cluster metrics that give a
probability of linkage to a single source?
• Perform phylogenetic analysis of epidemiologically confirmed
outbreaks.
I. Calculate SNP distance.
II. Examine tree structure.
• Do for many serovars of each species.
• This could be done relatively easily by freezer diving and HiSeq
runs.
• For SE SNP distances appear to be small (0-3 snps)
I. Based on 9 bonafide outbreaks from NY and MN.

16. NY-swgs1311
swgs1065
Large Western NY pattern 5 cluster
• 13 isolates collected over 10
months (0 to 6 snps distance)
• First isolates in fall 2013.
• February 2014, two distinct clades
form.
• Suggests bug has evolved
• Does this cluster represent 1, 2, or
3 sources?
NY-swgs1335
NY-swgs1347
NY-swgs1366
NY-swgs1305
swgs1217
7/05/14 Oneida
5/12/14 Ontario
2/6/14 Onondaga
NY-swgs1387_NEW
NY-swgs1360
6/22/14 Seneca
NY-swgs1339
6/11/14 Oswego
2/22/14 Oneida
2/21/14 Onondaga
12/12/13 Cattaraugus
9/25/13 Niagara
10/15/13 Monroe
swgs1224
2/22/14 Onondaga
NY-swgs1333
swgs1226
swgs1223
swgs1079
swgs1008
swgs1018
NY-swgs1355
7/22/14 Onondaga
6/3/14 Erie
1+snps+
NY-swgs1375_NEW

17. One person
two outbreaks
• GC-35 appears 6/9/14
• PFGE pattern 4
• Isolate from food a handler
• Total of 5 cases through 8/7/14
• GC-40 appears 6/30/14
• PFGE pattern 21
• isolate recovered from the same
food handler
JEGX01.0021++
JEGX01.0004

18. WGCA is better, but is it faster and cheaper?
metric( PFGE( WGCA(
TAT : extraction to
analysis
2 days 6+days+
Cost+ $69+ $294+
Technician+@me+ 8h+ 10h+
Ac@onable+clusters++ 3+nonEendemic+ 63+

19. We have created a two State Network
• Collaborating with Minnesota.
• Currently no informatics in house.
• We pull their sequences off Basespace.
• Run through our pipeline.

20. Tree from Merged
data
• Does pipeline
used to merge
the data affect
tree structure?
• Do sequence
metrics affect
merged tree
structure?
Travel+associated+

21. National Genomic Surveillance Machine
• State labs feed the machine by uploading sequences from isolates
received through surveillance.
• Federal and other support for reagents and equipment.
• NCBI to analyze the products of this machine and reports results to
state and federal agencies.

22. Current FDA Genome Trackr
network
State Health labs
• New York
• Florida
• Arizona
• Washington
• Minnesota
• Virginia
• Maryland
FDA labs
• 9 FDA field labs
• CFSAN - MOD1
• CFSAN - Wiley
• IEH (contracting lab)
International labs
• Mexico
• Ireland
• UK (FERA)
• Columbia
Contributors
• Turkey
• Brazil
• Italy

23. NCBI Pathogen Pipeline Salmonella tree
Travel associated

24. Expected Outcomes for WGS surveillance
• Laboratory
• Improve outbreak cluster detection.
• Clusters will be detected more rapidly and from fewer isolates.
• Epi
• Allow identification of clusters within endemic patterns.
• Solve more clusters.
• Public Health
• More efficient identification and removal of pathogen sources.

25. Challenges exist
• Creating a network.
• Increasing amounts of data.
• Metadata: how much should be public?
• In real time?
• What elements?
• Paying
• As sequencing technology and bioinformatics evolve:
• Need to maintain backward compatibility
• Transitioning:
• What to do first.
• Integration with serology and PFGE typing.

26. Standards I would like to see
• Pipeline quality and reproducibility.
• Tree quality and reproducibility.
• Probability metrics that a cluster is from a single source.

27. Summary
• WGS can improve surveillance activities and outbreak traceback.
• It is practical to develop network.
• We need standards.

28. • Cornell Acknowledgments
Martin Wiedmann
Henk den Bakker
• FDA
Eric Brown
Peter Evans
Marc Allard
Errol Strain
Ruth Timme
• Connecticut DOH
Stacey Kinney
John Fontana
• Minnesota DOH
David Boxrud
Angie Jones
Victoria Lappi
• Washington State DOH
Ailyn Perez-Osorio
Zhen Li
• Wadsworth Center Genomics Core
Matt Shudt
Zhen Zhang
Charles MacGowan
Melissa Leisner
Danielle Loranger
Mike Palumbo
Pascal LaPierre
• Kara Michell
• Wadsworth PulseNet Lab
Dianna Bopp
Deb Baker
Lisa Thompson
• NCBI
Bill Klimke
Martin Shumway