Subjunc

# 1) Standard paired-end RNA-seq alignment
subjunc \
  -i ref_index \
  -r sample_R1.fastq.gz \
  -R sample_R2.fastq.gz \
  -o sample.bam \
  -T 8

# 2) Coordinate-sorted BAM for downstream browsing/counting
subjunc \
  -i ref_index \
  -r sample_R1.fastq.gz \
  -R sample_R2.fastq.gz \
  -o sample.bam \
  -T 8 \
  --sortReadsByCoordinates

# 3) Broader junction discovery, including non-canonical events
subjunc \
  -i ref_index \
  -r sample_R1.fastq.gz \
  -R sample_R2.fastq.gz \
  -o sample.bam \
  -T 8 \
  --allJunctions

Recommended Workflow

1. Build a Subread index once and reuse it across the cohort.
2. Run subjunc on RNA-seq FASTQ with paired-end information if available.
3. Sort output if downstream tools or browsers expect coordinate order.
4. Count with featureCounts and inspect suspicious junction-rich loci separately.

Guardrails

• -i expects the pre-built index basename, not the original FASTA file.
• subjunc is RNA-oriented; use subread-align -t 1 for ordinary genomic DNA alignment.
• The default -m 1 is permissive; tighten thresholds if false junctions become a problem.
• The default maximum indel length is 5 bp; raise -I when longer indels are biologically expected.
• --allJunctions expands reporting scope and can increase noisy findings, so use it deliberately.