GenomeTrakr WGS Protocol Collection and Workflow for MiSeq
Ruth Timme, Maria Balkey, Julie Haendiges, Candace Hope Bias, Tina Lusk Pfefer
Disclaimer
Abstract
Here we have created a collection of all the protocols used for WGS using the MiSeq, in order, from sample extraction to NCBI submission.
This collection has three sections:
- Section 1: WGS Wet lab workflow for Illumina MiSeq
- Section 2: Dry lab workflow for sequence QC and NCBI submission - Direct Submission
- Section 3: Dry lab workflow for sequence QC and NCBI submission - PulseNet labs
Version history:
V3:
- Links to protocols updated with latest version,
- Added new protocol: DNA Extraction from Inoculated Broth for WGS using the Maxwell RSC Cultured Cells DNA Kit,
- Added new protocol: Querying for Bacterial Pathogen Genomic Data at NCBI.
Steps
WGS Wet Lab Workflow for Illumina MiSeq
DNA Extraction - Manual
Manual DNA Extraction using Qiagen DNeasy Blood and Tissue Kit
DNA Extraction - Automated
DNA Extraction from Inoculated Broth for WGS using the Maxwell RSC Cultured Cells DNA Kit
DNA Quantification
DNA Quantification using the Qubit Fluorometer
Library Preparation
Illumina DNA Prep (M) Tagmentation Library Preparation for use on an Illumina MiSeq Sequencer
Sequencing
Procedure for Operation and Maintenance of the Illumina MiSeq for Whole Genome Sequencing
Dry Lab Workflow for Sequence QC and NCBI Submission - Direct Submission
The following protocols are also included in a Springer Methods book chapter collection: Utilizing the Public GenomeTrakr Database for Foodborne Pathogen Traceback (protocols.io)
Check sequence quality:
Quality control assessment for microbial genomes: GalaxyTrakr MicroRunQC workflow
Populate BioSample AND SRA metadata templates:
Guidance for populating and validating GenomeTrakr metadata templates (BioSample and SRA)
Submit sequence and metadata to NCBI:
NCBI submission protocol for microbial pathogen surveillance
Querying data at NCBI
Querying for Bacterial Pathogen Genomic Data at NCBI
Update, retract, or replace your submissions at NCBI:
NCBI Bacterial Pathogen Data Curation Protocol: SOP for Editing GenomeTrakr Submissions
Dry Lab Workflow for Sequence QC and NCBI Submission - PulseNet Labs
Check sequence quality:
Follow the CDC's guidance for assessing QC in BioNumerics
Populate NCBI template:
***NOTE: For NCBI submission for fields not included in the BioNumerics Template, please remember to include the name of the laboratory sequencing the isolates and the surveillance effort name in the sequenced_by and project_name fields as described in Step 5 of this protocol.
Populating NCBI template for submissions using BioNumerics
Submit sequence and metadata to NCBI:
Follow the CDC's guidance for NCBI submissions through BioNumerics.
Query data at NCBI
Update, retract, or replace these records in NCBI databases, if necessary:
