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This tutorial is forked from vappiah/bacterial-genomics-tutorial, share with our lab co-worker as educational use, Thanks alot !

Download and install anaconda(version 3 recommended)

Add channels

conda config --add channels conda-forge\
conda config --add channels bioconda\
conda config --add channels daler\
conda config --add channels defaults\

Download the Analysis pipeline

git clone https://github.com/vappiah/bacterial-genomics-tutorial.git

Change directory to the dowloaded folder

cd bacterial-genomics-tutorial

Create conda environment.Packages are listed in the environment.yaml file.

conda env create -f environment.yaml

Download the polishing tool pilon

mkdir apps\
wget https://github.com/broadinstitute/pilon/releases/download/v1.23/pilon-1.23.jar -O apps/pilon.jar

Activate the analysis environment

source activate bacterial-genomics-tutorial

Add permission to all scripts

chmod +x *.{py,sh,pl}

Install python packages using pip

pip install -r pip-requirements.txt

TIME FOR ANALYSIS

Step 1: Download data.

./download_data.sh

Step 2: Perform QC on the raw reads

./qc_raw_reads.sh

Step 3: Trim reads using sickle

./trim_reads.sh

Step 4: Perform QC on the trimmed reads

./qc_trimmed_reads.sh

Step 5: Perform de novo assembly using spades

./assemble.sh

### Step 7: Perform QC for both raw assembly and polished assembly

./qc_assembly.sh


### Step 8: Generate draft genome by reordering contigs against a reference genome using ragtag\

./reorder_contigs.sh

Step 10: Check for antimicrobial resistance genes using abricate\

./amr.sh

Step 11: Annotate the draft genome using prokka

./annotate.sh

Step 12: Get some statistics on the annotation.

Features such as genes, CDS will be counted and displayed. The scripts requires you to specify the folder where annotations were saved . i.e. P7741 Python should be used to run that script

python get_annot_stats.py P7741_annotation P7741

Step 13: Generate dendogram using dREP\

./dendogram.sh

Step 14: Perform Pangenome Analysis using Roary.

Input files are gff (version 3 ) format. It is recommended to use prokka generated gff. So we generate the gffs for the files in the genome folder by reannotating with prokka. We use the get_genome_gffs script
./get_genome_gffs.sh

Then perform pangenome analysis\

./get_pangenome.sh

Step 15: Get gene summary for three of the organism. the default is P7741 Agy99 and Liflandii. Feel free to change it. A venn diagram will be generated(gene_count_summary.png)

python gene_count_summary.py P7741 Agy99 Liflandii pangenome/gene_presence_absence.csv

If you are working on a cluster you will want to combine the analysis results into a zip file for download and view locally. ./zip_results.sh

Step 16: Compare your draft genome with the other organisms in the genomes folder by generating circular structures for them . Use the tutorial here to guide you https://youtu.be/pobQgE4z-5Q

Step 17: Result interpretation

The result interpretation are available on my youtube video tutorial : https://youtu.be/S_sRo_85jhs

Now that you have been able to perform a bacterial comparative genome analysis. Its time to apply your skills on a real world data. Good luck and see you next time

Citation

Vincent Appiah, 2020. Bacterial Genomics Tutorial https://github.com/vappiah/bacterial-genomics-tutorial