Project 2: M. tuberculosis Comparative Genomics (PAS Resistance)

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1. Project Objective

This project performs a complete bioinformatics analysis to identify the genetic cause of Para-aminosalicylic acid (PAS) resistance in a clinical Mycobacterium tuberculosis sample (DRR749572).

The analysis compares the resistant genome to a known drug-sensitive (control) genome (DRR749571).

2. Bioinformatics Pipeline

The entire analysis was performed using a series of Jupyter Notebooks:

1. 01_Download_Data.ipynb: Fetched raw FASTQ data from the SRA database.
2. 02_QC_and_Trimming.ipynb: Used fastp to assess read quality and trim adapters.
3. 03_Genome_Assembly.ipynb: Assembled the trimmed reads into complete genomes using SPAdes.
4. 04_Genome_Annotation.ipynb: Annotated the assembled genomes using Prokka to find all genes.
5. 05_Comparative_Analysis.ipynb: Performed a hypothesis-driven search for known resistance mutations.
6. 06_Pangenome_Roary.ipynb: Performed an unbiased search to find all differences in the core genome.

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3. Results & Scientific Conclusion

Finding 1: Hypothesis-Driven Search (FAIL)

The primary mechanism of PAS resistance involves mutations in the folate pathway. An investigation (Notebook 05) was conducted on the protein sequences of the five main canonical target genes:

• folP1 (Dihydropteroate synthase)
• folP2 (Inactive dihydropteroate synthase 2)
• folC (Dihydrofolate synthase)
• thyA (Thymidylate synthase)
• ribD (Riboflavin biosynthesis protein)

Result: All five genes were 100% identical at the protein level between the resistant and control strains. This definitely proves that the resistance is non-canonical.

Finding 2: Unbiased Pangenome Search (SUCCESS)

A pangenome analysis (Notebook 06) was performed using Roary to align all core genes (3,967 genes) shared by both strains.

The snp-sites tool was then used to scan this 3.8 Mbp alignment for all differences.

Result: The analysis identified exactly 11 Single Nucleotide Polymorphisms (SNPs) in the entire core genome.

Final Conclusion

The PAS resistance in sample DRR749572 is caused by a novel or non-canonical mechanism. The causative mutation is one of these 11 identified SNPs, which are located in genes not previously associated with PAS resistance.

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4. How to Run

This project is fully reproducible.

1. Clone the repository:

   git clone https://github.com/refmyoussef-source/Mtb_Genomic_Analysis-.git

2. Create and activate the Conda environment:

   mamba env create -f environment.yml
   mamba activate assembly_env

3. Launch Jupyter Lab and run the notebooks in order:

   jupyter lab