Pipeline

HOMERun analyzes csRNA-seq data as a series of steps, each handled by an established bioinformatics tool and submitted as SLURM jobs.

1. Adapter trimming

Sequencing reads carry adapter sequence that must be removed, and csRNA/sRNA reads are size-selected (roughly 20–58 nt).

• Tool: HOMER homerTools trim (csRNA, sRNA); skewer for paired-end totalRNA

2. Alignment to the genome

Trimmed reads are mapped to a reference genome to determine where each read originated, including strand.

• Tool: STAR (default), HISAT2 (optional)
• Output: Aligned/<sample>.Aligned.out.sam

3. Tag directory creation

Alignments are converted into HOMER tag directories — position-sorted, depth-normalized read counts plus precomputed QC statistics.

• Tool: HOMER makeTagDirectory
• Built per replicate (leaf) and merged per assay (combo)

4. BedGraph generation

Genome-browser tracks are generated from the tag directories for visualizing signal along the genome.

• Tool: HOMER makeUCSCfile

5. TSS / TSR calling

Capped-RNA peaks are called to identify active transcription start regions, using sRNA as an input control and totalRNA as a stability reference.

• Tool: HOMER findcsRNATSS.pl
• Output: Species/TSS/<sample>.tss.txt

6. Quality control & reporting

Per-sample QC is written under Species/QC/<sample>/. Compact cross-sample A/C/G/T nucleotide-divergence heatmaps (PNG/SVG plus TSV matrices) are written directly under Species/QC/ for csRNA and sRNA.

7. Stability analysis

Called TSRs are classified as stable or unstable transcripts using the totalRNA reference (HOMER Log2Ratio vs. stableRNA).