Lab 10 — Genome Browsers and Alignment

Key concepts and tools

• Bowtie2 — splice-unaware short-read aligner, index building
• STAR — splice-aware RNA-seq aligner, index building
• samtools sort, samtools index
• BAM and BAI file format
• bamCoverage (deepTools) — BAM to bigWig conversion
• bigWig format, coverage tracks
• IGV — loading BAMs, bigWigs; paired-end read visualization
• Splice-aware vs. splice-unaware alignment: visual differences at exon junctions
• branch operator — routing samples to different processes

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You will build a Nextflow pipeline that aligns RNA-seq reads with two different aligners — Bowtie2 (splice-unaware) and STAR (splice-aware) — then sorts and indexes both BAM files and converts them to bigWig coverage tracks. Several modules are provided; you complete the samtools sort, samtools index, and bamCoverage modules and write the workflow that connects them.

Workflow tasks

1. Build a Bowtie2 index and a STAR index for human chr16 (hg38)
2. Align the bowtie2 sample with Bowtie2 and the star sample with STAR — use a channel operator to route each sample to the correct aligner
3. Sort and index both resulting BAM files with samtools
4. Generate a bigWig coverage track from each BAM with bamCoverage

Samples are subsetted for speed — run locally:

nextflow run main.nf -profile singularity,local

IGV

Open an SCC Desktop interactive session (OnDemand) to run the IGV desktop app. Load both BAM files and both bigWig tracks. Navigate to a gene with multiple exons and observe how reads from the STAR-aligned BAM span splice junctions while reads from the Bowtie2 BAM do not.

Pre-generated results are available at /projectnb/bf528/materials/lab07_template/visualization/ if your pipeline is still running.