Whole Genome Sequence Analysis | Bacterial Genome Analysis | Bioinformatics 101 for Beginners

Introduction

This tutorial provides a comprehensive guide to analyzing whole genome sequences of bacterial genomes using various bioinformatics tools. It is designed for beginners in bioinformatics and will walk you through each step of the analysis process, from data acquisition to visualization of results. Understanding bacterial genome analysis is crucial for fields such as microbiology, epidemiology, and biotechnology.

Step 1: Acquire Example Data

Access the source of the example data at the National Center for Biotechnology Information (NCBI)

• NCBI Article
• Download the relevant FASTQ files for your analysis.

Step 2: Set Up the Analysis Pipeline

• Install Anaconda on your system to manage the necessary software and libraries.

  For installation instructions, refer to

  • Linux Installation
  • MacOS Installation
• Clone the GitHub repository containing the analysis scripts:

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

Step 3: Quality Control of Sequencing Data

• Use FastQC to perform quality control on the downloaded FASTQ files:

  fastqc yourdata.fastq
  

• Review the FastQC output for quality metrics such as sequence quality scores, GC content, and duplication levels.

Step 4: Trim FastQ Reads

• Use Sickle to trim low-quality reads:

  sickle pe -f yourdata.fastq -t sanger -o trimmed_output.fastq
  

Step 5: Genome Assembly

• Utilize SPAdes for genome assembly:

  spades.py -1 forward_reads.fastq -2 reverse_reads.fastq -o assembly_output
  

• Ensure to check the assembly quality.

Step 6: Evaluate Genome Assembly

• Use QUAST to evaluate the quality of your assembled genome:

  quast.py assembly_output/contigs.fasta
  

Step 7: Reference Guided Scaffolding

• Employ RagTag for scaffolding the assembly against a reference genome:

  ragtag.py scaffold assembly_output/contigs.fasta reference_genome.fasta
  

Step 8: Genome Annotation

• Annotate the genome using PROKKA:

  prokka --outdir annotation_output --prefix your_genome_name assembly_output/contigs.fasta
  

Step 9: Multi Locus Sequence Typing

Conduct MLST analysis to identify sequence types

• Use the relevant gene targets and the corresponding tools for your organism.

Step 10: Antimicrobial Resistance Gene Detection

• Detect antimicrobial resistance genes using Abricate:

  abricate --db ncbi assembly_output/contigs.fasta
  

Step 11: Pangenome Analysis

• Perform pangenome analysis using Roary:

  roary -f pangenome_output -e -n *.gff
  

Step 12: Genome Comparison

• Visualize the results using BRIG:

  brig your_genome.fasta reference_genome.fasta
  

Conclusion

In this tutorial, you have learned how to analyze whole genome sequences of bacteria from data acquisition to visualization. Key tools discussed include FastQC, Sickle, SPAdes, QUAST, RagTag, PROKKA, Abricate, Roary, and BRIG. For further exploration, consider diving deeper into each tool’s documentation and experimenting with your datasets. This foundational knowledge in bioinformatics will pave the way for more advanced analyses in the future.