Bacterial Strain Typing Workflow

PMI Workflow – Bacterial Strain Typing Workflow

The bacterial strain typing workflow is intended to provide 16S rDNA sequencing and analysis of bacterial taxonomy for strains isolated from Populus root and rhizosphere samples within the PMI project.

Step 1 Experiment Information

This step includes general information about source of bacterial isolates, PI, experimenter, technician etc.

    Entered Date:
    Experiment Name:
    Technician/Experimenter
    Associated PIs:
    Origin and date of bacterial isolate:
    Comments:   Any extra information as required
    File: Upload Date

Step 2 Sample generation

This step provides information on bacterial isolate growth and sample generation.


    Entered Date:
    Technician/Experimenter:
    Bacterial sample generation:
    Sample Generated Date:
    Bacterial isolate stock location:
    Sample Generated Location:
    Sample Generated by/Received:
    Sample Received Date:
    Growth medium and volume:
    Comments:   Any extra information as required
    File: Upload Date (should include spreadsheet containing unique isoilate identifiers for samples generated)

Step 3 Cell lysis, DNA Extraction and PCR Amplification


This step should include details regarding method for lysis of cells and extraction of DNA(if needed) and PCR amplification of 16S rDNA from bacterial samples. 

    Cell lysis and DNA extraction
    Method / Kit 
    Date
    Technician/Experimenter
    Notebook reference
    Comments:
    Files:
    PCR amplification
    Date
    Technician/Experimenter
    Notebook reference
    Conditions
    Primer Set / Region:
    Amplicon Quantification
    Comments:
    Files:


Step 4 Sequencing

Sequencing of 16S rDNA amplicon and taxomonic assignment. This step should include details on sequencing method used and method used for taxonomic analysis of sequence results. This typically is done by uploading a tab delimited FASTA

    Sequencing 
    Date
    Technician/experimentor
    Instrument Type
    Reagent  Type
    Vendor Software
    Internal Standard(s)
    Primer Set 
    Comments:
    Files: 
    Excel spreadsheet containing summary report of sequencing results
    FASTA file of assembled sequences for each isolate
    Optional file: ABI sequence trace files
    Analysis
    Analysis Program Type
    Program Version
    Date
    Experimenter
    Comments:
    Files: RDP classifier output is taxonomic assignment with confidence scores in comma delimited text file