GenomeTrakr WGS Protocol Collection and Workflow for MiSeq
Ruth Timme, Julie Haendiges, Maria Balkey, 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
Associated protocols:
- Querying the NCBI database for GenomeTrakr data (protocols.io)
- NCBI data curation protocol - SOP for editing GenomeTrakr submissions (protocols.io)
Steps
Wet lab
WGS Wet lab workflow for Illumina MiSeq
DNA Extraction
Manual DNA Extraction using Qiagen DNeasy Blood and Tissue 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 - Direct Submission
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 GenomeTrakr metadata templates (BioSample and SRA)
Submit sequence and metadata to NCBI:
NCBI submission protocol for microbial pathogen surveillance
Update, retract, or replace these records in NCBI databases, if necessary:
NCBI data curation protocol - SOP for editing GenomeTrakr submissions (protocols.io)
Dry lab - PulseNet submission
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:
Populating NCBI template for submissions using BioNumerics v7.6
Submit sequence and metadata to NCBI:
Follow the CDC's guidance for NCBI submissions through BioNumerics.
Update, retract, or replace these records in NCBI databases, if necessary:
NCBI data curation protocol - SOP for editing GenomeTrakr submissions (protocols.io)