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Jamie Wasilenko, Ph.D.Eastern Laboratory Microbiology Characterization Branch
FSIS USDA Office of Public Health Science
USDA, Food Safety and Inspection Service
Whole Genome Sequencing at FSIS: Pipelines and Informatics
May 22, 2017
Food Safety and Inspection Service
Whole Genome Sequencing (WGS) in FSIS- Overview
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Whole Genome Sequencing Methodology in FSIS WGS highlights
Current status
Analytical tools
Application hqSNP analysis
AMR
Harborage
Concluding remarks
Food Safety and Inspection Service
FSIS Organizational Structure
Office of the Administrator
Office of Data Integration and Food
Protection (ODIFP)
Office of Field Operations
(OFO)
Office of Outreach, Employee
Education and Training (OOEET)
Office of Public Affairs
and Consumer Education (OPACE)
Office of Management
(OM)
Office of Investigation, Enforcement
& Audit (OIEA)
Office of Public Health
Science (OPHS)
Office of Policy and Program
Development (OPPD)
Includes Compliance and
Investigations Division (CID)
Includes Recall Management
and Technical Analysis Division (RMTAD)
Includes Applied Epidemiology Staff
(AES), Science Staff (SciS), and Food Safety Laboratories
Includes Congressional &
Public Affairs Staff (CPA)
Food Safety and Inspection Service
FSIS Eastern Laboratory and Characterization Branch
Office of Public Health and Science
Eastern Laboratory
Microbiology Characterization
Branch
Microbiology Screening
Branch Chemistry Branch Pathology Branch
Midwestern Laboratory
Western Laboratory
Laboratory Quality
Assurance Staff
Food Emergency Response Staff
Russell Research Center, Athens, GA
Our Branch – Staff of 17
Pulsed-field gel electrophoresis (PFGE)Performed on all isolates (Salmonella, Campylobacter, L.
monocytogenes, E. coli O157:H7, and non-O157 STEC)Analyze images and uploaded to CDC-PulseNet
Salmonella serotypingMolecular serotyping and/or NVSL traditional serotyping
Campylobacter speciationC. jejuni, C. lari, and C. coli
Antimicrobial Resistance ProfilesPerformed on all Salmonella isolates, Campylobacter, STEC,
NARMS Enterococcus and NARMS E. coli isolates
Whole genome sequencing
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Food Safety and Inspection Service
Microbial Characterization Tools to Support Food Safety
Food Safety and Inspection Service:
WGS in FSIS: Sources of Bacterial Cultures for WGS
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• PRJNA242847 - GenomeTrakr Project: USDA – FSIS (Salmonella)
• PRJNA215355 - GenomeTrakr Project: FDA (Listeria monocytogenes)
• PRJNA287430 - USDA-FSIS: Isolated strains of Campylobacter spp. genome sequencing
• PRJNA268206 - GenomeTrakr Project: USDA – FSIS Shiga toxin-producing E. coli (STEC)
• PRJNA292666 - FSIS NARMS Salmonella
• PRJNA292667 - FSIS NARMS Escherichia coli
• PRJNA292668 - FSIS NARMS Campylobacter
• PRJNA292669 - FSIS NARMS Enterococcus
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Food Safety and Inspection Service:
WGS in FSIS: NCBI BioProjects - Data related to a single initiative
4/2014• 2 MiSeqs in EL
7/2014
• Sequence Outbreak Isolates
• FDA uploads FSIS sequences to NCBI
5/2015
• EL uploads directly to NCBI
• 15 Lm isolates
10/2016
• Goal of 100% of FSIS isolates sequenced
• ~7,000 isolates sequenced and uploaded
3/2017• Lab-wide SOP for WGS
Food Safety and Inspection Service
Past Efforts: WGS – How did we get here?
Additional Key points: 7 Miseqs, hiring of three Public Health Specialist (Bioinformatics) positions
Food Safety and Inspection Service
WGS in FSIS: Timeline to WGS completion
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Sample shipped to Field Service Lab (FSL)
1 day
Characterization (PFGE, AST, serotyping, speciation)
6 days
DNA isolation and quantification
1 day
WGS library prep
1-2 days
Load/Run Miseq2-3 days
WGS data transfer, QC and upload
1 day
Isolation at FSLs4-7 days
Ship sample to MCB-EL
1 day
WGS Timeline - 16 Isolates
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DNA Extraction from clonal source (16 isolates)3 hours
Day 1 cont.
Day 1
DNA Quality Assessment and Quantitation (Qubit): 2-3 hours
Day 2
Quantify DNA and Library Prep: 3 hours
Day 2 cont.
Day 3
MiSeq Run times300 Cycle Kit-26 hours500 Cycle Kit- 36 hours
Day 3-5
Dilution of DNATagmentation, and Indexing: 3 hours
Indexed product cleanup: 2 hours
Day 6 Data Transfer and Analysis3-8 hours
Combine indexed DNA and load into cartridge: 2 hoursDay 3 cont.
Food Safety and Inspection Service
WGS in FSIS: WGS Methodology
Food Safety and Inspection Service:
WGS in FSIS: Post Sequencing Data Analysis and QC
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MiSeq Run Metrics
Cluster Density, Pass Filter, Q30% Illumina Sequence Analysis Viewer (SAV) – Percent Index
and File Size
Assembly
CLC Genomics Ver. 8 de novo Assembly (fasta) SPAdes version 3.7
BioSample and
SRA upload
Create BioSample for each isolate Link FASTQ files & metadata to BioSample Upload to NCBI via Aspera command-line
Quality Trimming
and Coverage
Quality trimming (Trimmomatic) Average quality assessment-CGPipeline Nucleotide balance Pre and post-trim coverages calculated
QualityControl
Python scripts and BLASTdb for organism check BLASTdb for Campylobacter speciation Kraken for detection of contamination
Gene Identification
ResFinder BLASTdb Virulence BLASTdb Escherichia coli/Salmonella serotyping BLASTdb
Food Safety and Inspection Service:
WGS in FSIS: Downstream analysis tools
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Analysis Tool Source
Identification of Acquired Antibiotic Resistance Genes
• Local ResFinder
• BLASTdb
• http://www.genomicepidemiology.org
Identification STEC Virulence Genes
• VirulenceFinder
• BLASTdb
• stx and eae subtypes
• http://www.genomicepidemiology.org
MLST Identification- 7 Genes • Local BLASTdb • http://www.genomicepidemiology.org
• http://www.pubmlst.org
Identification of Contamination • Kraken • http://ccb.jhu.edu/software/kraken/
Whole Genome MLST • BioNumerics version 7.5/7.6
• CDC-PulseNet
• CDC-PulseNet Calculation Engine and Applied Maths
• http://www.applied-maths.com
SNP Calling and Phylogeny Inference
• Lyve-SET 1.14f
• CFSAN SNP Pipeline
• NCBI Pathogen Detection
• https://github.com/lskatz/lyve-SET
• https://github.com/CFSAN-Biostatistics/snp-pipeline
• http://www.ncbi.nlm.nih.gov/pathogens
Food Safety and Inspection Service
WGS in FSIS: WGS Data Flow
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Food Safety and Inspection Service
WGS in FSIS: WGS Data Flow
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Product/Source type (Ready to eat product, raw meat/poultry, environmental swab, etc.)
Year sample was collected
State where sample was collected
Subtyping information Salmonella – serovar
Escherichia coli– O-group
Campylobacter – species
Metadata and sequence data is immediately available for upload to NCBI
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Food Safety and Inspection Service
WGS in FSIS: Minimal Metadata Shared on NCBI
Methods of further investigating possible relationships between isolates of interest
High Quality SNP analysis
Whole genome MLST
BLAST based detection of genes (AMR genes)
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Food Safety and Inspection Service:
WGS in FSIS: Tools used for further analysis
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Applications of WGS at FSIS: Lm HarborageFood Safety and Inspection Service
WGS in FSIS: ‘WGS and PFGE’ – Listeria Experience (Ex-1)
WGS and PFGE comparison
High quality SNPs wgMLST
Same PFGE pattern: WGS agreement 0-2 SNPs 0-7 allele differences
Same PFGE pattern: WGS exclusion 34-45 SNPs 29-33 allele differences
Different PFGE: WGS inclusion 0-3 SNPs 0-5 allele differences
WGS and PFGE results are generally in agreement WGS can sometimes exclude isolates from a group
with the same PFGE pattern or include isolates in a group with different PFGE patterns
In the examples below inclusion or exclusion considered all information including sample metadata (establishment, isolation date, etc.)
Note the trend between the SNP and wgMLST approaches
FSIS is also currently exploring use of WGS as a tool to understand potential harborage of L. monocytogenesin establishments
FSIS works collaboratively with FDA in dual-jurisdiction establishments that produce both FDA and FSIS-regulated productsWhen one agency identifies potential harborage through
bacterial characterization of Listeria isolates (PFGE and/or WGS), information is shared to inform a collaborative regulatory response within the establishment
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Applications of WGS at FSIS: Lm HarborageFood Safety and Inspection Service
WGS in FSIS: Potential use in harborage
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Applications of WGS at FSIS: Lm HarborageFood Safety and Inspection Service
WGS in FSIS: Listeria Experience (Ex-3) - hqSNP Analysis
Lyve-SET 1.14f 90% read support, 20x coverage, 5bp flanking, reference: FSIS1505523 (27 contigs).
SNP matrices were produced using CFSAN SNP pipeline. Trees were assembled in MEGA7 using the Maximum Likelihood method based on the Tamura-Nei model with FSIS1605523 (27 contigs) as the reference.
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Applications of WGS at FSIS: Lm HarborageFood Safety and Inspection Service
WGS in FSIS: Listeria Experience (Ex-3) - wgMLST
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Applications of WGS at FSIS: Lm HarborageFood Safety and Inspection Service
WGS in FSIS: Outbreak Investigations
For all outbreak investigations we coordinate with our public health partners (CDC, FDA, state health departments)
FSIS food isolates included in analyses are chosen using epidemiological data from AES, CDC, and state epidemiologists, and based on subtyping information of historical strains from FSIS sampling programs
Analysis methods used include high quality SNP (hqSNP) analysis and whole genome MLST (wgMLST)
Analysis methods used for regulatory decision-making are those developed and agreed upon by our GenFS partners (CDC, FDA, NCBI)
WGS is currently done concurrently with PFGE and other subtyping methods, no decisions have been made solely on WGS
Food Safety and Inspection Service:
WGS in FSIS: Caveats and Considerations
Currently there are no policies in place for FSIS usage of WGS data to make WGS decisions
How close is close? The is no established metric that states a minimum number of SNPs or allele differences equates to isolates having a common ancestry
Lack of nomenclature, there is no easily communicated WGS “pattern name” that is analogous to PFGE pattern name
Food Safety and Inspection Service:
How is FSIS addressing caveats and considerations?
Currently working on Federal register notice Will state FSIS’ intent to use WGS regulatory decisions and explain
rationale Will engage stakeholders and allow for comments
FSIS plans to hold a Public Meeting regarding implementation of WGS
FSIS is a member of Interagency Collaboration on Genomics and Food Safety (Gen-FS) Federal partners: CDC, FDA, NCBI and FSIS Agencies with public health and regulatory focus Discussions is ongoing to include USDA-ARS and USDA-APHIS in Gen-FS
Dr. Glenn Tillman
Dr. Mustafa Simmons
Eastern Laboratory Staff
Public Health Partners
Food Safety and Inspection Service:
Acknowledgements