National Center for Emerging and Zoonotic Infectious Diseases

PulseNet Updates: Transitioning to WGS for Reference Testing and Surveillance

Kelley Hise, MPH

Enteric Diseases Laboratory BranchDivision of Foodborne, Waterborne and Environmental Diseases

PulseNet/OutbreakNet West Coast Regional MeetingFebruary 5, 2019

Overview Transition Timeline Updates on Conversions/Certifications Sequencing Prioritization Turn Around Times Data Analysis Workflow with the National Databases Combined Organism Databases

Transition TimelineUpdates on Conversions and Certifications

Dates for PulseNet’s Transition to WGS as the Gold Standard for Foodborne Surveillance

January 15, 2018

Listeria

October 1, 2018

Campylobacter

March 15, 2019Salmonella,

STEC, Shigella

WA

OR

MT

WY

COUT

AZ

ID

CA NV

ND

SD

KS

NE

TX

NMOK

MN

MO

IA

LA

AR

WI

IN

KY

MI

OHIL2

TN

GAALMS

SC

FL

PA

NC

WV VA

DEMD

NJ

ME

VT

NYNH

MACT

AK

HI

RI

Modified: February 1, 2019

PR

Converted to BioNumerics 7.6:31 states35 labs

PulseNet, WGS and Enhanced Epidemiological CapacityConverted to BioNumerics 7.6

OutbreakNet Enhanced or FoodCORE

DC

Area Laboratories

PulseNet Central

NYC

CA2

CAOC

CASC

LAC

HU

NVLV

IL

WGS Analysis Certified:5 states5 labs

Converted to BioNumerics 7.6 and WGS analysis certified for Listeria, Salmonella, Escherichia and Campylobacter

Not WGS wet lab certified

NYAG

NJEP

FNE

USDA/FSIS (EL)

USDA (MWL)

USDA/FSIS (WL)

FDA

Conversion Tips You MUST clean your local databases

– DATES: must be in correct format– KEYS: no spaces at the end– BUNDLES: permanent bundles should be deleted or moved to a different

location– PLUGINS: all plugins must be deactivated. The MLVA plugin is known to cause

an issue when converting.– LIBRARIES: all libraries should be deleted– COMPARISONS: saved comparisons will be lost in the conversion

For more detailed info: SharePointPulseNet DocumentsDatabaseCleaning Guidelines

Conversion Tips Local IT support should be readily available the week of your

conversion Review the Prep instructions Review training documents on SharePoint so you are ready to upload

PFGE patterns as soon as you have converted (WGS tools to be added in March)– SharePointPulseNet DocumentsWGSPHL Upgrade to BioNumerics

v7.6BN7 PFGE Data Management

Call/email PulseNet with questions related to conversion Once converted, email pulsenet@cdc.gov to let them know and

request analysis certification information

Analysis Certifications: Request after Conversion PNQ08 has been updated WGS Analysis Certification is available for Escherichia,

Salmonella, Listeria, and Campylobacter using BioNumerics v 7.6

Receive: (1) Certification set assignment A, B or C (i.e. Listeria Certification Set_A) and (2) Instructions for accessing fastq files, associated metadata, a bundle file and an analysis submission template via the PulseNetQA FTP site

Analysis Certifications: Tips Do not read too much into the quality metrics threshold tables

– No need to list every possible reason a sequence should be repeated

• Pay attention to the metrics in Red on the table in PNQ08-6• Metrics in black type are important and can provide information

but aren’t required for data to be uploaded to PulseNet– Use metrics listed and not those for wet lab certifications (i.e.

cannot find median insert size in BioNumerics)– Look at NCBI submission presentation and SOP to understand NCBI

metadata requirements

Sequencing Prioritization and Turn Around Times

Sequencing Prioritization (as of March 15, 2019) Listeria and STEC: sequence all isolates Salmonella: sequence all isolates if possible

– prioritize isolates with cluster codes (while PFGE remains)– random sequencing, e.g. every other or every third– as requested by CDC and/or epi

Campylobacter and Shigella: sequence all isolates, but prioritize other organisms first*

*unless specifically funded by other projects, like FoodNet

Turn Around Times (TATs) Starting January 1, 2019 TAT will be calculated from the date

the isolate was received (or recovered) in the PHL to the date of upload to the national database

Day 1 is date of receipt of a culture; for CIDTs, day 1 is not until an isolate is recovered

Should be 7 working days or less for WGS Keep track of local TATs

– Track steps along the way to determine areas for improvement

Turn Around Times: Calculate in BioNumerics

1. Select entries to calculate TAT

2. Click on clock at top of screen

3. Define parameters

NOTE • Upload_Date is not available• PulseNet_UploadDate is populated in BN7.6 upon upload• Suggest moving contents of Upload_Date to PulseNet_UploadDate for

entries prior to conversion

Data Analysis Workflow with the National Databases

2. Save generated sequence files locally, on BaseSpace, or external hard drive

1. Sequence isolates using PulseNet Key number

File naming format:Key-LabID-M###-YYMMDD

Data Analysis Workflow with National Database

Raw Sequence Data Private Raw

Sequence Storage

Public Raw Sequence

Data Storage

Organism-specific Database

Reference ID Database

PHL

3a. Link sequence data to Reference ID database by PulseNet Key name3b. Submit data to calculation engine (CE) for denovo assembly, species identification (Genus, Species – by ANI)3c. Verify quality3d. Export entries

4a. Import entries from Reference ID4b. Add demographic information for entries4c. Submit to the CE* for allele calls and genotyping results (serotype, AST, virulence)4d. Verify quality and upload to national database (WGS id automatically assigns)4e. Upload raw data sequence reads to NCBI4f. Perform surveillance in BioNumerics

PulseNet National

Databases

Updated 10/23/2018*CE: Calculation Engine

Calculation Engine (CE)

Calculation engine built to be highly customizable with easy integration of both custom-made and open source code

CE Store: server on the CE that states upload their data to Offers temporary storage of

sequences QA/QC, trimming, mapping, SNP

detection, allele detection

Calculation Engine

CDC

RefID Database to Organism-Specific Database Select sequences to submit to the

CDC’s calculation engine (CE) and retrieve the de novo assemblies and basic QC metrics

Once assemblies received, resubmit to CE and get back the taxonomic identification

De novo assemblies, QC metrics and taxa ID can then be exported and imported into the organism-specific database based on the genus/species identified. Either create new entry or link to previously imported entry (i.e. PFGE already done).

3d. Export de novo assemblies, QC

metrics, taxa ID to correct organism-specific database

Organism-specific Database

Reference ID Database

3b and c. Submit raw reads, and retrieve assembly with basic QC

metrics

PHL

Allele Databases

Calculation Engine

CDC

Submission to the Calculation Engine

Select sequences for analysis Choose from a list of algorithms to select

for analysis (figure) Submit assemblies and raw reads to the

CDC calculation engine

Allele Databases

Calculation Engine

CDC

Organism-specific Database

4c. Submit sequence data for allele calls

and genotyping results

PHL

Analyzed Results in BioNumerics User retrieves jobs from

calculation engine Allele calls/additional quality

metrics are imported into user database, and includes predicted: Serotype Resistance Virulence

Allele Databases

Calculation Engine

CDC

Organism-specific Database

Retrieve allele calls after submission—Predicted Serotype,

Resistance, Virulence

PHL

Upload to the PulseNet National Database Authenticate to PulseNet firewall Select entries and analyzed data to

upload to PulseNet national databases User can search national database for

close matches to uploaded sequence data and download things like outbreak codes and allele codes

PulseNet National

Databases

Organism-specific Database

CDC

4e. Upload allele calls and metadata

Download allele code, outbreak code, etc.

PHL

Submission to NCBI Can create and save templates for

upload of biosample, sequence metadata, and fastq files to NCBI

Can import NCBI-assigned ids back into user database (e.g. NCBI accession and SRR numbers)

Organism-specific Database

PHL

Public Raw Sequence Data

Storage

Upload raw sequence data with minimal metadata

For more information, contact CDC1-800-CDC-INFO (232-4636)TTY: 1-888-232-6348 www.cdc.gov

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

Questions?

Telephone: 404-639-4558E-mail: PulseNet@cdc.gov Web: www.cdc.gov/pulsenet

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