Report 03 - Genome Browser Visualization: RNA-seq Analysis of Plasmodium chabaudi

A practical guide to genome visualization using IGV and UCSC Genome Browser, developed for the "Bioinformatics II" module in the Genomic Sciences program at UNAM Juriquilla.

Overview

This project demonstrates a complete workflow for visualizing next-generation sequencing data, including:

• Loading and exploring reference genomes and annotations
• Processing RNA-seq alignments (SAM/BAM conversion, sorting, indexing)
• Visualizing read alignments with IGV
• Interpreting coverage tracks, splice junctions, and alignment features
• Exploring Mus musculus hepatic ChIP-seq data

Data

Plasmodium chabaudi (malaria parasite)

• Reference genome: PccAS_v3_genome.fa (indexed with .fai)
• Genome annotation: PccAS_v3.gff3 (GFF3 format with gene features)
• RNA-seq samples: MT1 and MT2 (paired-end FASTQ files)

Mus musculus (mouse)

• Reference genome: mm10.fa (UCSC assembly)
• ChIP-seq data: mus_musculus_CEBPA_liver_ERR005132.fastq.gz (hepatic tissue)

Pipeline

Step	Tool	Description
Genome indexing	hisat2-build	Build HISAT2 index from reference FASTA
Read alignment	hisat2	Splice-aware alignment of RNA-seq reads
Format conversion	samtools view	Convert SAM to BAM format
Sorting	samtools sort	Sort alignments by genomic coordinates
Indexing	samtools index	Generate BAM index (.bai) for fast access
Visualization	IGV	Interactive genome visualization

Key Commands

Genome indexing and alignment

# Index the reference genome
hisat2-build PccAS_v3_genome.fa PccAS_v3_hisat2.idx

# Align paired-end reads (RNA-seq with intron support)
hisat2 --max-intronlen 10000 -x PccAS_v3_hisat2.idx \
       -1 MT1_1.fastq -2 MT1_2.fastq -S MT1.sam

BAM processing

# Convert SAM to BAM
samtools view -bS MT1.sam > MT1.bam

# Sort by genomic coordinates
samtools sort MT1.bam -o MT1_sorted.bam

# Index for visualization
samtools index MT1_sorted.bam

Dependencies

# Load modules (cluster environment)
module load hisat2/2.0.0-beta
module load samtools/1.9

Software requirements:

• HISAT2 – Splice-aware aligner for RNA-seq
• SAMtools – SAM/BAM manipulation
• IGV – Integrative Genomics Viewer (local installation)

Usage

1. Prepare the reference: Index the genome with hisat2-build
2. Align reads: Run HISAT2 with appropriate parameters for RNA-seq
3. Process alignments: Convert, sort, and index BAM files
4. Visualize in IGV:
   • Load reference genome: Genomes → Load Genome from File
   • Load annotations: File → Load from File (GFF3)
   • Load alignments: File → Load from File (sorted BAM)

Note: BAM files and their .bai index files must be in the same directory for IGV to load them correctly.

Output

• MT1_sorted.bam / MT2_sorted.bam: Coordinate-sorted alignments
• *.bam.bai: BAM index files for efficient visualization
• IGV tracks showing:
  • Coverage depth across the genome
  • Splice junctions (strand-specific coloring)
  • Individual read alignments with variants highlighted

IGV Visualization Features

Track Type	Description
Coverage	Gray histogram showing read depth; colored bars indicate variants (>20% frequency)
Splice junctions	Red (+ strand) and blue (− strand) arcs connecting exons
Alignments	Individual reads with insertions (purple) and deletions (−) marked
Annotations	Gene models with exons (boxes) and introns (lines); arrows indicate strand

Author

Axel Rodriguez Perez
Genomic Sciences, UNAM · March 2023