Salmonella and Escherichia Lyophilized cultures: Salmonella and Escherichia 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.

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) and incubate at 37°C overnight in aerobic conditions. It is recommended to plate in duplicate to ensure adequate growth.

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

Check the BAPs; 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

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 plate as appropriate and incubate at 37°C overnight in aerobic conditions. On the next day check BAP plate and proceed as step 4.

Campylobacter Campylobacter Lyophilized cultures:

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.

Re-suspend the lyophilized cells with 1ml of sterile phosphate-buffered saline . Mix gently to produce a uniform suspension. Plate 100 μl of this suspension onto a non-selective BHIRB plate plate and incubate microaerobically at 42°C for 48 hr. It is recommended to plate in duplicate to ensure adequate growth.

Check the BHIRB plate for purity and pick an isolated colony and streak it onto a fresh BHIRB plate.

Incubate microaerobically at 42°C overnight. Use the growth from this plate to make DNA extracts of the PT strains. Transfer culture to fresh medium and incubate at 42°C overnight; this will ensure that the same culture can be retested, if necessary.

** - Each isolate for the proficiency testing exercise shall be processed as any routine isolate according to laboratory guidelines.

* PulseNet Labs will process these isolates according to PulseNet guidelines.

Perform DNA extraction according to lab’s normal workflow described on GenomeTrakr and/or PulseNet SOPs.

Manual DNA Extraction using Qiagen DNeasy Blood and Tissue Kit

** The sequencing run for the PT exercise shall be loaded as any routine run following established loading requirements.

** Control reference strains shall be included in the run when there is not enough isolates to fill a sequencing run.

Perform library preparation according to lab’s normal workflow described on GenomeTrakr and/or PulseNet SOPs. Isolates must be processed along with routine isolates to meet the genome loading requirements for the sequencing cartridge described in protocol Illumina DNA Prep (M) Tagmentation Library Preparation for use on an Illumina MiSeq Sequencer V.3.

Illumina DNA Prep (M) Tagmentation Library Preparation for use on an Illumina MiSeq Sequencer

If you don’t have enough isolates for your sequencing run, please run reference control isolates in the sequencing run.

Quantify DNA according to lab’s normal workflow described on GenomeTrakr and/or PulseNet SOPs.

DNA Quantification using the Qubit Fluorometer

Sequence DNA libraries according to lab’s normal workflow described on GenomeTrakr and/or PulseNet SOPs.

Procedure for Operation and Maintenance of the Illumina MiSeq for Whole Genome Sequencing

Sequencing sample sheets must be filled out according to Table 1.

Sample_ID or Sample_Name: Include in these fields the strain identifiers as it is indicated in Table 1, do not modify these IDs. You will also find this identifier in the vial of the lyophilized culture.

A	B	C	D
Sample_ID	Sample_Name	Project	Description
	ESP24-4709	PR0506_2024_Proficiency_Testing_Exercise	Escherichia coli
SAP24-0369	SAP24-0369	PR0506_2024_Proficiency_Testing_Exercise	Salmonella enterica
SAP24-0005	SAP24-0005	PR0506_2024_Proficiency_Testing_Exercise	Salmonella enterica
CAP24-7320	CAP24-7320	PR0506_2024_Proficiency_Testing_Exercise	Campylobacter jejuni

Table 1: PT strain identifiers for Sequencing Sample Sheet (PT isolates processed on a sequencing run with historical and/or routine isolates)

NextSeq 1000/2000 and MiSeq systems running Windows 10 (MCS v4 and up) have only Sample_ID available in the sequencing sample sheet, make sure to include the strain identifier as indicated in Table 1, columns Sample_ID and Sample_Name.

Project: Please fill out the project field with the project identifier PR0506_2024_Proficiency_Testing_Exercise.

Description: For your use only, we do not track this field. Organism names might be included in this field.

PulseNet labs will fill out the sequencing sample sheet according to PulseNet guidelines.

Perform quality control of sequencing data according to lab’s normal workflow described on GenomeTrakr and/or PulseNet SOPs.

Quality control assessment for microbial genomes: GalaxyTrakr MicroRunQC workflow

Data transfer

After checking the quality of your records, transfer the data with acceptable quality according to GenomeTrakr guidelines via BaseSpace or share drive.

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, click share button, then select send invitation.

• Enter the email address for the FDA team (gnometrakr@fda.hhs.gov) under -invite a collaborator- option, and then click Add Collaborator.

• Click Share the associated Project as well.

• Click Save Settings. Your run will be automatically shared with the GenomeTrakr team.

Sharing a project in BaseSpace without sharing a run

• Click the Projects tab in the Illumina BaseSpace website .

• Select the project ( PR0506_2024_Proficiency_Testing_Exercise) that you would like to share with the GenomeTrakr team.

• Click the share project button, then select send invitation.

• 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.

Labs inside the FDA network must share the the sequencing files by transferring the sequencing run folder to the isilon storage drive.

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 “2024 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