Two-pass mode

Two-pass alignment improves recovery of reads spanning novel splice junctions — those not present in the GTF (or when no GTF is supplied). rustar-aligner runs both passes in a single command, matching STAR’s --twopassMode Basic.

How it works

1. Pass 1 aligns all reads (or a subset, see --twopass1readsN) against the original index. Splice junctions discovered during this pass are filtered and collected.
2. The discovered junctions are inserted into a per-run on-the-fly junction database.
3. Pass 2 re-aligns all reads against the augmented index. Reads that previously soft-clipped over an unannotated junction now stitch through it.

The pass-1 junctions are filtered the same way as STAR: by motif (canonical GT/AG vs non-canonical), unique-mapping support, distance to other junctions, and intron length vs read count. Defaults are STAR-compatible.

Basic command

rustar-aligner \
  --genomeDir /path/to/genome_index \
  --readFilesIn reads_1.fq.gz reads_2.fq.gz \
  --readFilesCommand zcat \
  --twopassMode Basic \
  --outSAMtype BAM SortedByCoordinate \
  --outFileNamePrefix sample_

Combining with a GTF

You can use two-pass mode together with --sjdbGTFfile. Annotated junctions from the GTF are loaded into the index alongside the pass-1 discoveries; a junction in both gets the higher-confidence treatment.

rustar-aligner \
  --genomeDir /path/to/genome_index \
  --readFilesIn reads_1.fq.gz reads_2.fq.gz \
  --readFilesCommand zcat \
  --sjdbGTFfile gencode.v45.gtf \
  --twopassMode Basic \
  --outSAMtype BAM SortedByCoordinate \
  --outFileNamePrefix sample_

Limiting pass 1

For very large datasets you can limit pass 1 to a subset of reads:

--twopass1readsN 1000000

This processes the first million reads in pass 1, then runs pass 2 on the full input. The default (-1) uses all reads in both passes.

Junction filter parameters

The defaults match STAR. The most relevant filters:

• --outSJfilterOverhangMin — min overhang per motif category [noncanon, GT/AG, GC/AG, AT/AC]. Default 30 12 12 12.
• --outSJfilterCountUniqueMin — min unique-mapping reads per motif. Default 3 1 1 1.
• --outSJfilterCountTotalMin — min total reads per motif. Default 3 1 1 1.
• --outSJfilterIntronMaxVsReadN — junction is filtered when intron length exceeds the threshold for its read count. Default 50000 100000 200000 (1, 2, 3+ supporting reads).

See the CLI parameters reference for the full list.

When to use it

Use two-pass mode when:

• Working with a species or sample where the GTF annotation is incomplete
• You expect novel junctions (cancer transcriptomes, non-model organisms, single-cell)
• You want to recover the maximum number of cross-junction reads

Skip it when:

• Speed matters more than novel junction sensitivity
• You have a high-quality, complete GTF and don’t need novel discovery

Output

Two-pass mode writes the same set of files as a single-pass run. The SJ.out.tab reflects the final pass-2 set of junctions used during alignment.