Apr 09, 2025
Version 2
Standardized Sequencing Guidance for Microbiological Control Strains V.2
- Ruth Timme1,
- Tina Pfefer2,
- Candace Hope Bias3,
- Maria Balkey1
- 1US Food and Drug Administration;
- 2US FDA;
- 3US FDA-CFSAN
- GenomeTrakrTech. support email: genomeTrakr@fda.hhs.gov
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/v2Version created by Ruth Timme
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: April 09, 2025
Protocol Integer ID: 126449
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.
V2: updated metadata guidance in Table 1.
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:
FDA 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
NAME
GenomeTrakr WGS Protocol Collection and Workflow for MiSeqCREATED 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
NAME
Quality control assessment for microbial genomes: GalaxyTrakr MicroRunQC workflowCREATED 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.
| A | B | |
| Attribute Name | Guidance and examples | |
| sample_name | Isolate ID provided by the laboratory | |
| bioproject_accession | For submitters connected to the GenomeTrakr network, use PRJNA981165. | |
| isolate_name_alias | other strain IDs for the isolate, separated by “; ” | |
| strain | Use the offical control strain name here. For ATCC strains, enter a space between ATCC and the ID: ATCC ##### | |
| culture_collection | If the strain originates from an institutional culture collection (such as ATCC), please enter the catalog number using the format ATCC:12345. Leave this field blank if the strain is personal, agency-owned, or not from a curated collection. | |
| reference_material | Enter the term "laboratory control strain" here. | |
| organism | Enter the full binomial taxonomic name, e.g. "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 | Enter the term "laboratory control strain" here | |
| source_type | Choose the picklist term "Other" | |
| samp_collect_device | BLANK | |
| purpose_of_sampling | choose the picklist term "Research" | |
| project_name | For GenomeTrakr collaborating labs, use the term "GenomeTrakr" here. For LFFM labs, include the funding year, e.g. GenomeTrakr; LFFM-FY5. | |
| IFSAC_category | BLANK | |
| lat_lon | BLANK | |
| serotype | BLANK | |
| serovar | BLANK | |
| sequenced_by | Lab name performing the sequencing | |
| description | Purcuase/acquisition date of control strain = YYYY-MM-DD |
Table: Metadata guidelines for control strain sequencing
NCBI submission:
Protocol
NAME
NCBI submission protocol for microbial pathogen surveillanceCREATED BY
Ruth Timme
BioProject: For labs collaborating with GenomeTrakr on this project, please use PRJNA981165 (FYI: these sequences will not appear in Pathogen Detection).