Mar 24, 2025

Public workspaceStandardized Sequencing Guidance for Microbiological Control Strains

DOI
dx.doi.org/10.17504/protocols.io.14egn9pd6l5d/v1
  • 1US Food and Drug Administration;
  • 2US FDA;
  • 3US FDA-CFSAN
  • GenomeTrakr
    Tech. support email: genomeTrakr@fda.hhs.gov
Ruth Timme
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DOI: dx.doi.org/10.17504/protocols.io.14egn9pd6l5d/v1
Protocol Citation: Ruth Timme, Tina Pfefer, Candace Hope Bias, Maria Balkey 2025. Standardized Sequencing Guidance for Microbiological Control Strains . protocols.io https://dx.doi.org/10.17504/protocols.io.14egn9pd6l5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 13, 2025
Last Modified: March 24, 2025
Protocol Integer ID: 124351
Keywords: GenomeTrakr, Quality control, control strains, reference database
Abstract
This protocol provides guidance for sequencing laboratory control strains and submitting those data to NCBI. The BioProject established for this project aims to capture the genetic/genomic diversity of control strains being used in public health laboratories. This resource will support a new quality control step for identifying rare but significant false positives in labs processing foodborne pathogen isolates, helping to reduce food waste and unnecessary product disposal.
The Standardized Sequencing Guidance for Control Strains protocol provides an SOP for generating high-quality whole-genome sequence data from bacterial control strains used in public health, academic, and industry laboratories sequencing bacterial isolates of foodborne pathogens.

By systematically sequencing and depositing these control strain genomes into a publicly accessible reference database at the National Center for Biotechnology Information (NCBI), this initiative aims to document the genomic diversity of strains used across laboratories globally. The resulting dataset will support new quality control measures to detect and mitigate rare but significant false-positive sample test results that could lead to unwarranted public health actions, such as food recalls or product disposal.

This protocol establishes a standardized approach to control strain sequencing, ensuring consistency across laboratories. The resulting database will serve both public health agencies as as well as industry laboratories conducting food testing.
Scope for control strains: All actively used microbiological laboratory control strains are eligible for inclusion in this project. See Attached Table for a list of control strains used in FDA laboratories:

Download FDA controls strains.xlsxFDA controls strains.xlsx

Laboratory Methods:
Control strain isolates should be processed according to standard GenomeTrakr or PulseNet laboratory procedures.

GenomeTrakr WGS workflow protocol collection provided here:
Protocol
GenomeTrakr WGS Protocol Collection and Workflow for MiSeq
NAME
GenomeTrakr WGS Protocol Collection and Workflow for MiSeq
CREATED BY
Tina Pfefer

Quality Assessment

Check that WGS sequence data meet the GenomeTrakr thresholds listed in the following protocol. Laboratories are welcome to use MicroRunQC or any other equivelent QC pipeline:
Protocol
Quality control assessment for microbial genomes: GalaxyTrakr MicroRunQC workflow
NAME
Quality control assessment for microbial genomes: GalaxyTrakr MicroRunQC workflow
CREATED BY
Ruth Timme

Metadata guidance:

Use the latest version of the One Health Enteric BioSample package and adhere to the guidelines in the following table:

Populate the CORE package attributes only.
AB
Attribute NameGuidance and examples
sample_nameIsolate ID provided by the laboratory
bioproject_accession PRJNA981165
isolate_name_alias other strain IDs for the isolate, separated by “; ”
strain ATCC #####
culture_collection ATCC:#####
reference_material laboratory control strain
organism example "Salmonella enterica"
collected_by Laboratory name where control strain is being used
collection_date Collection date for sampling the control strain
cult_isol_date BLANK
geo_loc_name USA:State of laboratory location
isolation_source laboratory control strain
source_type Other
samp_collect_device BLANK
purpose_of_sampling Research
project_name example "GenomeTrakr"
IFSAC_category BLANK
lat_lon BLANK
serotype BLANK
serovar BLANK
sequenced_by Lab name performing the sequencing
description Acquisition date of control strain = YYYY-MM-DD
Table: Metadata guidelines for control strain sequencing
NCBI submission:
Protocol
NCBI submission protocol for microbial pathogen surveillance
NAME
NCBI submission protocol for microbial pathogen surveillance
CREATED BY
Ruth Timme
BioProject: For labs collaborating with GenomeTrakr on this project, please use PRJNA981165 (FYI: these sequences will not appear in Pathogen Detection).