# 2023 GenomeTrakr Proficiency Testing Exercise (PulseNet Harmonized) V.7
## Goal/Experiment:
The goal of this proficiency testing exercise is to provide standardized guidelines for GenomeTrakr (GT) laboratories to process isolates, generate sequencing data, and report results for the 2023 GenomeTrakr Proficiency Testing exercise. The exercise aims to ensure consistent and accurate detection of foodborne pathogens using Whole Genome Sequencing (WGS).
### Authors:
Maria Balkey¹, Ruth Timme¹, Julie Haendiges¹, Tina.Pfefer¹
¹ U.S. Food and Drug Administration
## Abstract:
This Standard Operating Procedure (SOP) outlines guidelines for processing the isolates for the 2023 GenomeTrakr (GT) Proficiency Testing exercise. It is applicable to all GenomeTrakr labs participating in the 2023 GenomeTrakr Proficiency Testing exercise (PulseNet Harmonized).
### Version Updates:
- **v5:** Added links to GalaxyTrakr_PT_exercise_report_2022.xlsx
- **v6:** added 2023 PT strains; edited Steps 1, 2, 4, and 5 to include references to growth of Listeria on BHIA plates; Updated Tables in Steps 7.1 and 8.1 with sample information for 2023 PT strains.
- **v7:** Section 9 and Section 11: deleted file GalaxyTrakr_PT_exercise_report_2023.xlsx; uploaded new file version GalaxyTrakr_PT_exercise_report_2023-v2.xlsx
### Proficiency Testing Isolates:
The FDA GenomeTrakr program will ship the following proficiency testing isolates in April 2023:
| Strain ID | Organism |
| ------------- | ----------------------- |
| ESP23-5286 | Escherichia/Shigella |
| ESP23-9201 | Escherichia/Shigella |
| SAP23-3307 | Salmonella |
| LMP23-6714 | Listeria |
Completion of the entire proficiency test entails the following:
- GT participating laboratory generates sequencing data (fastq files) using the PT strains provided by CDC through FDA-CFSAN-WGS Program-GT in accordance with GT and/or PulseNet SOPs.
- Populate sample sheet according to 2023 GT Proficiency Testing exercise (PulseNet Harmonized) SOP.
- Submission of sequencing records to the appropriate project on BaseSpace or Isilon according to GT SOPs.
- By participating in the 2023 GT Proficiency Testing exercise (PulseNet Harmonized), GT labs provide consent to use the PT exercise data in subsequent analysis and manuscript publications. Participants will be acknowledged for their contribution on any publication that might require processing data from the 2023 GT PT exercise.
## Materials
### Materials Needed:
- Sterile sturdy forceps
- 1 ml pipetman
- 1 ml sterile pipet tips
- 1 µl and/or 10 µl sterile inoculating loop
### Reagents Needed:
- Trypticase Soy + 5% Sheep Blood Agar plates (BAP) or equivalent media
- BHIA plates
- Sterile reagent grade water or Phosphate Buffered Saline (0.01M PBS; pH 7.4)
- BHI broth
- 70% isopropyl alcohol
## Safety Warnings
- **Biological Safety Warning:** _Escherichia/Shigella_, _Salmonella_, and _Listeria_ strains are considered Level 2 biological agents by the U.S. Department of Health and Human Services. Use appropriate precautions when handling the vial or culture. Carry out laboratory work in a biological safety cabinet when applicable to ensure aseptic conditions and personal safety.
## Before Start Instructions:
There are four sections in this protocol:
1. Culture preparation of lyophilized isolates.
2. Sequencing
3. Data Transfer
## Culture Preparation
### 1. Salmonella, Escherichia/Shigella, and Listeria Lyophilized cultures:
#### Day 1:
1. Document the isolate number(s) and the lyophilized date(s) for your records. Wipe the aluminum cover and outside of the vial with isopropyl alcohol. Using sturdy forceps, aseptically remove the aluminum cover and rubber stopper from the vial containing the lyophilized culture. Wipe the outside of the rubber stopper and neck of the vial with isopropyl alcohol before removing the stopper.
2. Re-suspend the lyophilized cells with 1 ml of sterile reagent grade water. Allow to stand for a few minutes and/or mix gently to produce a uniform suspension. Plate 10 µl of this suspension onto a blood agar plate (BAP) (or BHIA plate for Listeria) and incubate at 37°C overnight in aerobic conditions. It is recommended to plate in duplicate to ensure adequate growth.
3. Add the rest of the suspension to 5 mL of BHI broth and incubate the culture overnight at 37°C in aerobic conditions.
#### Day 2 and 3:
4. Check the BAPs and BHIA plates; if the culture appears pure, pick an isolated colony, and streak it on a fresh plate; incubate at 37°C overnight in aerobic conditions. Use the growth from this plate to make DNA templates of the PT strains. Transfer culture to fresh medium and incubate at 37°C overnight; this will ensure that the same culture can be retested, if necessary.
#### Optional:
5. If plates don't show bacterial growth, prepare a new plate by taking a loop from the BHI overnight culture (prepared at step 3), streak it on a BAP or BHIA plate as appropriate and incubate at 37°C overnight in aerobic conditions. On the next day check BAP or BHIA plate and proceed as step 4.
## Sequencing
### 6. Perform DNA extraction, library preparation and sequencing according to lab's normal workflow described on SOPs posted at GenomeTrakr protocols.io:
- [Manual DNA Extraction Using Qiagen DNeasy Blood & Tissue Kit](https://www.protocols.io/view/manual-dna-extraction-using-qiagen-dneasy-blood-an-81wgqb391qvk/v1)
- [Procedure for Operation and Maintenance of the Illumina MiSeq](https://www.protocols.io/view/procedure-for-operation-and-maintenance-of-the-ill-rm7vz8z52wx1/v1)
- [Illumina DNA Prep (M)-Tagmentation Library Preparation](https://www.protocols.io/view/illumina-dna-prep-m-tagmentation-library-preparati-x54v9m7ezg3e/v2)
- [DNA Quantification Using the Qubit Fluorometer](https://www.protocols.io/view/dna-quantification-using-the-qubit-fluorometer-81wgbp3x3vpk/v1)
### PulseNet Labs:
- Will process isolates according to PulseNet Guidelines.
- Isolates must be processed exactly as any routine isolate would be processed in the laboratory.
### 7. Sequencing sample sheets must be filled out according to Table 1:
#### 7.1 Sample_ID:
Include in this field the values from the Sample_ID column of Table 1,*do not modify these IDs*. You will also find this identifier in the vial of the lyophilized culture.
| Sample_ID | Sample_Name | Project | Description |
| ------------ | ----------------------| -----------------------------------------| ------------|
| ESP23-5286 | ESP23-5286-M0XXXX-20230424 | PR0403_2023_Proficiency_Testing_Exercise | Escherichia/Shigella |
| ESP23-9201 | ESP23-9201-M0XXXX-20230424 | PR0403_2023_Proficiency_Testing_Exercise | Escherichia/Shigella |
| SAP23-3307 | SAP23-3307-M0XXXX-20230424 | PR0403_2023_Proficiency_Testing_Exercise | Salmonella |
| LMP23-6714 | LMP23-6714-M0XXXX-20230424 | PR0403_2023_Proficiency_Testing_Exercise | Listeria |
#### Non PulseNet Labs:
NextSeq 1000/2000 and MiSeq systems running Windows 10 (MCS v4 and up) have only Sample_ID available in the sequencing sample sheet, populate the Sample_ID column with the values in the Sample_ID column of Table 1.
### 7.2 Sample_name:
#### Non PulseNet Labs:
- Fill out this field according to example provided in column Sample_Name of table 1. Include the isolate identifier, instrument ID (M0XXXX where "XXXX" corresponds to the instrument identifier) and run start date. (e.g. ESP23-5286_M01001-20230424).
#### PulseNet Labs:
- Include the isolate identifier, lab ID, Instrument ID (M0XXXX where "XXXX" corresponds to the instrument identifier) and run start date. (e.g. ESP23-5286-GA-M01001-20230424).
### 7.3 Project:
- Please fill out the project field with the project identifier PR0403_2023_Proficiency_Testing_Exercise.
### 7.4 Description:
- For your use only, we do not track this field. Organism names might be included in this field.
## Replicates
### 8. Are you running more than one set of PT isolates in a run?
- **YES:** Proceed to Step 8.1
- **NO:** Proceed to Step 9.
### 8.1 Modify Isolate identifiers (IDs at vial of lyophilized culture) to create unique identifiers for PT replicated isolates by adding suffixes such as: "_2" or "_3" to isolate identifiers.
#### Non-PulseNet Laboratories:
If you choose to run replicates, the sample sheet must contain unaltered isolate identifiers for each PT strain in the Sample_name field. Identifiers in the sample_ID field must include the isolate identifier, replicate, instrument Id and start run date.
| Sample_ID | Sample_Name | Project | Description |
| ----------------------- | ----------- | ----------------------------------- |-----------------------|
| ESP23-5286-1-M0XXXX-20230424 | ESP23-5286 | PR0403_2023_Proficiency_Testing_Exercise | Escherichia/Shigella |
| ESP23-9201-1-M0XXXX-20230424 | ESP23-9201 | PR0403_2023_Proficiency_Testing_Exercise | Escherichia/Shigella |
| SAP23-3307-1-M0XXXX-20230424 | SAP23-3307 | PR0403_2023_Proficiency_Testing_Exercise | Salmonella |
| LMP23-6714-1-M0XXXX-20230424 | LMP23-6714 | PR0403_2023_Proficiency_Testing_Exercise | Listeria |
...
#### PulseNet Laboratories:
- Identifiers in the sample_ID and sample_Name fields must include the isolate identifier, replicate, lab ID, instrument Id and start run date.
| Sample_ID | Sample_Name | Project | Description |
| --------------------------------- | ------------------- | ----------------------------------- |-----------------------|
| ESP23-5286_1_GA_M0XXXX_20230424 | ESP23-5286_1_GA_M0XXXX_20230424 | PR0403_2023_Proficiency_Testing_Exercise | Escherichia/Shigella |
| ESP23-9201_1_GA_M0XXXX_20230424 | ESP23-9201_1_GA_M0XXXX_20230424 | PR0403_2023_Proficiency_Testing_Exercise | Escherichia/Shigella |
| SAP23-3307_1_GA_M0XXXX_20230424 | SAP23-3307_1_GA_M0XXXX_20230424 | PR0403_2023_Proficiency_Testing_Exercise | Salmonella |
| LMP23-6714_1_GA_M0XXXX_20230424 | LMP23-6714_1_GA_M0XXXX_20230424 | PR0403_2023_Proficiency_Testing_Exercise | Listeria |
...
### 8.2 Project:
- Please fill out the project field with the project identifier PR0403_2023_Proficiency_Testing_Exercise.
### 8.3 Description:
- For your use only, we do not track this field.
## Quality of sequencing run
### 9. Data analysis results must be reported to GenomeTrakr in the spreadsheet included in the attachment.
- Check the quality of your sequencing records by following the SOP [Assessing sequence quality in GalaxyTrakr](https://). Note that Shigella isolates will be predicted as Escherichia coli with PubMLST scanning of contigs. The result is acceptable. Report your findings in the spreadsheet GalaxyTrakr_PT_exercise_report_2023-v2.xlsx, sheet MicroRunQC.
- Run the tool [ShigaTyper](https://) for Shigella genoserotyping. Report your findings in the spreadsheet GalaxyTrakr_PT_exercise_report_2023, sheet ShigaTyper.
- Run the tool [SeqSero2](https://) for Salmonella serotyping. Report your findings in the spreadsheet GalaxyTrakr_PT_exercise_report_2023, sheet SeqSero.
## Data Transfer
### 10. Data Transfer
After checking the quality of your records, transfer the data and associated QC results to GenomeTrakr.
#### 10.1 Sharing a run in BaseSapce
- Click the Runs tab in the Illumina BaseSpace website.
- Select the run that you would like to share with the GenomeTrakr team.
- Go to the summary tab and click at the share button.
- Enter the email address for the FDA team (gnometrakr@fda.hhs.gov), and then click Add Collaborator.
- Click Save Settings. Your run will be automatically shared with the GenomeTrakr team.
#### 10.2 Sharing a project in BaseSapce
- Click the Projects tab in the Illumina BaseSpace website.
- Select the project (PR0403_2023_Proficiency_Testing_Exercise) that you would like to share with the GenomeTrakr team.
- Click the share project button.
- Enter the email address for the FDA team (gnometrakr@fda.hhs.gov), and then click Add Collaborator.
- Click Save Settings. Your project will be automatically shared with the GenomeTrakr team.
#### 10.3 Labs inside the FDA network
- Must share the sequencing files by transferring the sequencing run folder to the isilon storage drive.
### 11. PT Exercise Completion Notification
- Notify GenomeTrakr of your completion of the PT exercise by sending an email to: gnometrakr@fda.hhs.gov. The subject line should include “2023 WGS Proficiency Testing_YourLabName”, attach sequencing sample sheet and include the following information in the body of the email:
- Run name:
- Sequenced by:
- Results submitted by:
- MiSeq ID:
- Flow cell ID:
- SOP Protocol: (Select one of the options)
- PulseNet SOP
- GenomeTrakr SOP
#### Non-PulseNet Laboratories, please include
- GalaxyTrakr_PT_exercise_report_2023-v2.xlsx in the attachments of the notification email.
#### PulseNet Laboratories
- Must send the 2023 Proficiency Testing exercise data and results to PulseNet.
### 12.
00:00:00
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