method.md

Methods Overview:

1. Data Collection:

   • Obtain Clostridioides difficile genome sequences and annotations (FASTA, GFF) from public databases.
   • Acquire RNA-Seq datasets under different growth conditions.

2. RNA-Seq Preprocessing:

   • Perform quality control (FastQC).
   • Trim low-quality reads and adapters (Trimmomatic).
   • Align reads to the reference genome (Bowtie2/Hisat2).

3. Operon Prediction:

   • Use Rockhopper for transcription unit identification.
   • Run COSMO to integrate gene co-expression patterns and predict operon structures.

4. Validation & Analysis:

   • Compare predicted operons with known databases.
   • Analyze differential gene expression to infer operon regulation.

5. Interpretation & Reporting:

   • Identify operons linked to antibiotic resistance and metabolic pathways.
   • Summarize findings and visualize operon structures.