TITLE: 2023 GenomeTrakr Proficiency Testing exercise (PulseNet Harmonized)
AUTHORS: Maria  Balkey, Ruth Timme, Julie Haendiges, Tina.Pfefer

[DESCRIPTION]
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.



This SOP outlines guidelines on how to process the isolates for the 2023 GenomeTrakr (GT) Proficiency Testing exercise.

This SOP is applicable to all GenomeTrakr labs participating in the 2023 GenomeTrakr Proficiency Testing exercise (PulseNet Harmonized).

The FDA GenomeTrakr program will ship the following proficiency testing isolates in April 2023.

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 to 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.



[BEFORE_START]
There are four sections in this protocol:

Section 1:  Culture preparation of lyophilized isolates.
Section 2:  Sequencing
Section 3:  Data Transfer


[STEPS]
SECTION: Culture Preparation
1. Salmonella,  Escherichia/Shigella, and Listeria Lyophilized cultures:

Day 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.
SECTION: Culture Preparation
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.
SECTION: Culture Preparation
3. Add the rest of the suspension to 5 mL of BHI broth and incubate the culture overnight at 37°C in aerobic conditions.
SECTION: Culture Preparation
4. Day 2 and 3

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.
SECTION: Culture Preparation
5. Optional

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.
SECTION: Sequencing
6. 
Perform DNA extraction, library preparation and sequencing according to lab’s normal workflow described on SOPs posted at GenomeTrakr protocols.io.

https://www.protocols.io/view/manual-dna-extraction-using-qiagen-dneasy-blood-an-81wgbp31qvpk/v1
https://www.protocols.io/view/procedure-for-operation-and-maintenance-of-the-ill-rm7vz8z52vx1/v1
https://www.protocols.io/view/illumina-dna-prep-m-tagmentation-library-preparati-x54v9m7ezg3e/v2
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.

SECTION: Sequencing
7. Sequencing sample sheets must be filled out according to Table 1.
SECTION: Sequencing
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_IDSample_NameProjectDescription  LMP20-6735  LMP20-6735_"XX"PR0379_2020_Proficiency_Testing_ExerciseListeria monocytogenes  LMP20-6494  LMP20-6494_"XX"PR0379_2020_Proficiency_Testing_ExerciseListeria monocytogenes  SAP20-8099  SAP20-8099_"XX"PR0379_2020_Proficiency_Testing_ExerciseSalmonella enterica  SAP20-6399  SAP20-6399_"XX"PR0379_2020_Proficiency_Testing_ExerciseSalmonella enterica

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.

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, follow PulseNet guidelines to populate Sample_ID.  Include the isolate identifier, lab ID, Instrument ID and run start date. (e.g. ESP23-5286-GA-M01001-20230424). 
SECTION: Sequencing
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). 

SECTION: Sequencing
7.3. Project:  Please fill out the project field with the project identifier PR0403_2023_Proficiency_Testing_Exercise.
SECTION: Sequencing
7.4. Description: For your use only, we do not track this field.  Organism names might be included in this field. 
SECTION: Sequencing
8. Replicates 

Are you running more than one set of PT isolates in a run?

YES: proceed to Step 8.1
NO: proceed to Step 9.

If you don’t have enough isolates for your sequencing run, you could run PT isolates multiple times. Because MiSeq does not allow duplicate sample_IDs, please fill out the sequencing sample sheet using the instructions included in Step 8.1. See Table 2 (Non-PulseNet) and 3 (PulseNet) for examples.  Only one set of files shall be shared with GenomeTrakr.
SECTION: Sequencing
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    ESP22-5191-1-M0XXXX-20220324  ESP22-5191  PR0393_2022_Proficiency_Testing_Exercise  Shigella    SAP22-0236-1-M0XXXX-20220324  SAP22-0236  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    SAP22-0692-1-M0XXXX-20220324  SAP22-0692  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    SAP22-1300-1-M0XXXX-20220324  SAP22-1300  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    ESP22-5191-2-M0XXXX-20220324  ESP22-5191  PR0393_2022_Proficiency_Testing_Exercise  Shigella    SAP22-0236-2-M0XXXX-20220324  SAP22-0236  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    SAP22-0692-2-M0XXXX-20220324  SAP22-0692  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    SAP22-1300-2-M0XXXX-20220324  SAP22-1300  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    ESP22-5191-3-M0XXXX-20220324  ESP22-5191  PR0393_2022_Proficiency_Testing_Exercise  Shigella    SAP22-0236-3-M0XXXX-20220324  SAP22-0236  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    SAP22-0692-3-M0XXXX-20220324  SAP22-0692  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    SAP22-1300-3-M0XXXX-20220324  SAP22-1300  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    ESP22-5191-4-M0XXXX-20220324  ESP22-5191  PR0393_2022_Proficiency_Testing_Exercise  Shigella    SAP22-0236-4-M0XXXX-20220324  SAP22-0236  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    SAP22-0692-4-M0XXXX-20220324  SAP22-0692  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    SAP22-1300-4-M0XXXX-20220324  SAP22-1300  PR0393_2022_Proficiency_Testing_Exercise  Salmonella  
  
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    ESP22-5191_1_GA_M0XXXX_20220324  ESP22-5191_1_GA_M0XXXX_20220324  PR0393_2022_Proficiency_Testing_Exercise  Shigella    SAP22-0236_1_GA_M0XXXX_20220324  SAP22-0236_1_GA_M0XXXX_20220324  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    SAP22-0692_1_GA_M0XXXX_20220324  SAP22-0692_1_GA_M0XXXX_20220324  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    SAP22-1300_1_GA_M0XXXX_20220324  SAP22-1300_1_GA_M0XXXX_20220324  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    ESP22-5191_2_GA_M0XXXX_20220324  ESP22-5191_2_GA_M0XXXX_20220324  PR0393_2022_Proficiency_Testing_Exercise  Shigella    SAP22-0236_2_GA_M0XXXX_20220324  SAP22-0236_2_GA_M0XXXX_20220324  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    SAP22-0692_2_GA_M0XXXX_20220324  SAP22-0692_2_GA_M0XXXX_20220324  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    SAP22-1300_2_GA_M0XXXX_20220324  SAP22-1300_2_GA_M0XXXX_20220324  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    ESP22-5191_3_GA_M0XXXX_20220324  ESP22-5191_3_GA_M0XXXX_20220324  PR0393_2022_Proficiency_Testing_Exercise  Shigella    SAP22-0236_3_GA_M0XXXX_20220324  SAP22-0236_3_GA_M0XXXX_20220324  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    SAP22-0692_3_GA_M0XXXX_20220324  SAP22-0692_3_GA_M0XXXX_20220324  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    SAP22-1300_3_GA_M0XXXX_20220324  SAP22-1300_3_GA_M0XXXX_20220324  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    ESP22-5191_4_GA_M0XXXX_20220324  ESP22-5191_4_GA_M0XXXX_20220324  PR0393_2022_Proficiency_Testing_Exercise  Shigella    SAP22-0236_4_GA_M0XXXX_20220324  SAP22-0236_4_GA_M0XXXX_20220324  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    SAP22-0692_4_GA_M0XXXX_20220324  SAP22-0692_4_GA_M0XXXX_20220324  PR0393_2022_Proficiency_Testing_Exercise  Salmonella    SAP22-1300_4_GA_M0XXXX_20220324  SAP22-1300_4_GA_M0XXXX_20220324  PR0393_2022_Proficiency_Testing_Exercise  Salmonella  
  


SECTION: Sequencing
8.2. Project: Please fill out the project field with the project identifier PR0403_2023_Proficiency_Testing_Exercise
SECTION: Sequencing
8.3. Description: For your use only,  we do not track this field. 
SECTION: Sequencing
9. Quality of sequencing run

-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. 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   , sheet MicroRunQC.

- Run the tool ShigaTyper Shigella genoserotyping. Report your findings in the spreadsheet GalaxyTrakr_PT_exercise_report_2023, sheet ShigaTyper.

- Run the tool SeqSero2 for Salmonella serotying. Report your findings in the spreadsheet GalaxyTrakr_PT_exercise_report_2023, sheet SeqSero.




SECTION: Data Transfer
10. Data transfer

After checking the quality of your records, transfer the data and associated QC results to GenomeTrakr. 
SECTION: Data Transfer
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.



SECTION: Data Transfer
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.
SECTION: Data Transfer
10.3. 
Labs inside the FDA network must share the the sequencing files by transferring the sequencing run folder to the isilon storage drive.  
SECTION: Data Transfer
11. PT exercise completion notification

Notify GenomeTrakr of your completion of the PT exercise by sending an email to: genometrakr@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:

a. Run name:
b. Sequenced by:
c. Results submitted by:
d. MiSeq ID:
e. Flow cell ID:

Select one of the options

f. SOP Protocol: 
    - PulseNet SOP  
    - GenomeTrakr SOP

Non-PulseNet Laboratories, please include      in the attachments of the notification email. 

PulseNet Laboratories must send the 2023 Proficiency Testing exercise data and results to PulseNet. 
SECTION: Data Transfer
12.