GenomeTrakr: Data Submission and Analysis

Transcription

1 GenomeTrakr: Data Submission and Analysis Ruth Timme and Hugh Rand Center for Food Safety and Applied Nutrition U.S. Food Drug Administration IFSH Whole Genome Sequencing for Food Safety Symposium SEPTEMBER 28-30, 2016 Chicago, Illinois

2 Support for Good Decision Making Quality control on data submission Sequence data analyses Publicly available data (CFSAN SNP Pipeline) Internal data SOP for conducting and interpreting analysis Validation for analysis pipeline Datasets Simulation pipeline Case studies, Building experience, Publishing it Proficiency testing (wet and dry)

3 Quality control on data submission CFSAN Robust sample tracking with Laboratory Information Management System Average sequence coverage Sequence quality (Fall 2016) NCBI Sequence quality Contamination Mixed samples Taxonomic ID (correct species?) 3

4 Sequence analysis: CFSAN SNP Pipeline

5 Interpretation of SNP Distances How close are isolates? No single threshold for all species/types Rough, conservative guides are used to classify a match Inclusion Inconclusive Exclusion Results are critically evaluated and not used blindly Is the cluster distinct from other isolates in the tree? Do the isolates form a unique cluster w/ >= 95% bootstrap support? Moving toward addressing all recommendations in PCAST report Forensic Science in Criminal Courts: Ensuring Scientific Validity of Feature-Comparison Methods

6 Validation: benchmark datasets Benchmarks allow: Evaluation of method performance Guidance on improvement of methods Standards for assessing reproducibility of results Bolster use of results for regulatory action

7 Benchmark Outbreak Datasets US Federal Agency collaboration: CDC, FDA, NCBI, USDA Species Isolates Outgroups Reference Outbreak E. Three coli WGS/Epi retrospective 10 outbreaks: Listeria monocytogenes Salmonella enterica Campylobacter jejuni Simulated data

8 Easy download at GitHub downloaddataset.pl <dataset_template.csv> Files for each dataset Epi data, in/out of outbreak, recommended reference. Fastq files annotated assemblies Known tree Validated SNPs (coming soon)

9 TreeToReads Validation: Simulated data Download here: EJ McTavish, J Pettengill, S Davis, H Rand, E Strain, M Allard, R Timme. TreeToReads - a pipeline for simulating raw reads from phylogenies. Accepted. BMC Bioinformatics.

10 Test the effect of different parameters and analysis methods Data: Sequence coverage (5x, 10x, 20x, etc) NGS read length (150bp, 250bp, etc) Amount of sequence divergence (total number of SNPs or variable sites) Tree shape (length of node leading to OB) Clumpiness of SNP distribution Analysis: SNP-based methods reference vs. ref-free SNP collection Distance of reference genome Effects of phylogenetic inference method? NJ, MP, ML. Maximum likelihood model misspecification? Alternate analysis methods (wgmlst, Kmer)

11 Case Studies: L. mono samples from 18 facilities Facility Product Cheese 10 2 Hot Peppers 18* 11 3 Ice Cream 4 4 Ice Cream 19 5 RTE Beans RTE Meals RTE Meals RTE Meals RTE Meals 3 10 RTE Meals Seafood 1* 3 12 Seafood Soft Cheese Soft Cheese 6 15 Soft Cheese Soft Cheese Soy Sauce Sprouts 35 * Product

12 Within vs Between Clone SNPs Facility Product n # Clones IntraSNP InterSNP 1 Cheese NA 2 Hot Peppers NA 3 Ice Cream Ice Cream RTE Beans > RTE Meals > RTE Meals NA 8 RTE Meals > RTE Meals NA 10 RTE Meals > Seafood > Seafood , > Soft Cheese NA 14 Soft Cheese NA 15 Soft Cheese Soft Cheese > Soy Sauce NA 18 Sprouts NA

13 SNPs - Year to Year, 10 facilities Facility Product Year to Year 2 Hot Peppers 21 5 RTE Beans 6 6 RTE Meals 8 7 RTE Meals 11 8 RTE Meals > Seafood > Seafood 4 15 Soft Cheese 8 16 Soft Cheese > Soy Sauce 6

14 Proficiency testing: GenomeTrakr 2014, 2015: Each lab in the GT network sequenced the same set of 8 strains. CFSAN PT analysis returned. Manuscript in preparation GMI (yearly since 2013) 2016 PT has wet and dry lab components 2016 PT includes K. pneumonia, L. mono, C. jejuni, E. coli PulseNet/GenomeTrakr harmonized PT Early 2017

15 CFSAN PT analysis:

16 How Far Along Are We? Quality control on data submission Sequence data analyses Publicly available data (CFSAN SNP Pipeline) Internal data SOP for conducting and interpreting analysis Validation for analysis pipeline Datasets Simulation pipeline Case studies, Building experience, Publishing it Proficiency testing (wet and dry)

17 Acknowledgements WGS Standards and Analysis workgroup (CDC/FDA/NCBI/FSIS) FDA: Ruth Timme, Hugh Rand, Steven Davis CDC: Lee S. Katz, Eija Trees, Heather A. Carleton NCBI: Richa Agarwala, Martin Shumway, Bill Klimke FSIS: Mustafa Simmons, Glenn Tillman, Philip Bronstein, Stephanie Defibaugh-Chávez Other contributors: Errol Strain James Pettengill Joe Baugher Emily Jane McTavish Maria Hoffmann Maria Sanchez 17