Chimeric detection

A chimeric alignment is a read whose two halves map to different genomic locations — different chromosomes, the same chromosome with an unrealistically large gap, or the same chromosome but on opposite strands. These are the candidate evidence for gene fusions, large-scale structural variants, and circular RNA back-splicing.

rustar-aligner uses STAR’s two chimeric detectors, ported as written; which one runs depends on --chimMultimapNmax.

Enabling chimeric detection

Chimeric detection is off by default (--chimSegmentMin 0). Enable it by setting a minimum chimeric segment length — STAR’s recommended starting value is 12:

rustar-aligner \
  --genomeDir /path/to/genome_index \
  --readFilesIn reads_1.fq.gz reads_2.fq.gz \
  --readFilesCommand zcat \
  --chimSegmentMin 12 \
  --outSAMtype BAM SortedByCoordinate \
  --outFileNamePrefix sample_

Higher values (e.g. 20) produce fewer, more confident calls; lower values (e.g. 10) are more sensitive but noisier.

What gets detected

Both detectors work on the read’s window transcripts, the alignments stitching produced before any filtering. For paired-end reads these are whole-fragment transcripts (mate1 | RC(mate2)), so one side of a chimera can be a complete mate pair.

• Default (--chimMultimapNmax 0): STAR’s chimericDetectionOld. The best linear alignment is one segment, and the detector looks for the single best partner that covers the rest of the read. A chimera is reported only if it is clearly better than the runner-up (--chimScoreSeparation), and at most one per read.
• --chimMultimapNmax N > 0: STAR’s chimericDetectionMult. Every pair of window transcripts is tried and re-scored after the junction is placed, and all chimeras within --chimMultimapScoreRange of the best are reported, up to N (more than N reports none). The junction file gains six columns: number of chimeras, maximum possible score, best non-chimeric score, this chimera’s score, best chimeric score, and whether the mates were merged.

When the junction falls inside a read, its position is chosen by STAR’s scan over the overlap, preferring GT/AG or CT/AC motifs; when the two mates of a pair lie on either side of it, the junction type is -1.

Output formats

Set --chimOutType to control the output. Multiple values are allowed.

Junctions (default)

--chimOutType Junctions

Writes a <prefix>Chimeric.out.junction file with one row per chimeric junction. The 14-column format matches STAR’s; tools like Arriba and STAR-Fusion consume it directly.

WithinBAM

--chimOutType WithinBAM [HardClip|SoftClip]
--outSAMtype BAM Unsorted        # or SortedByCoordinate; WithinBAM needs BAM output

Writes the chimera into the main BAM in place of the read’s normal alignment, as STAR does: one segment as a normal record and the other as a supplementary record (FLAG 0x800), with SA tags pointing at each other. The supplementary segment is hard-clipped by default (HardClip) or soft-clipped (SoftClip). For paired-end reads a segment that covers both mates is written as a normal pair; when the mates lie on either side of the junction, both are written as normal records with no supplementary. NM is added to the output attributes, as STAR does. This is the input Arriba recommends.

Because these reads are written as chimeras, STAR does not count them as uniquely or multi-mapped in Log.final.out, and they do not contribute to SJ.out.tab or gene counts; rustar-aligner does the same.

Mixed output

--chimOutType Junctions WithinBAM

Writes both the junction file and the chimeric BAM records.

Tuning parameters

The most useful chimeric parameters:

• --chimSegmentMin — minimum chimeric segment length (also enables/disables detection).
• --chimScoreMin — minimum total chimeric alignment score. Default 0.
• --chimScoreSeparation — minimum score gap between the chosen chimeric pair and the next-best alternative. Default 10.
• --chimJunctionOverhangMin — minimum bases on each side of the chimeric junction. Default 20.
• --chimMainSegmentMultNmax — main segment can multimap up to this many loci. Default 10.
• --chimScoreJunctionNonGTAG — score penalty for non-canonical chimeric junctions. Default -1.

See the CLI parameters reference for the rest.

Output columns

The Chimeric.out.junction file has 14 tab-separated columns (STAR-compatible):

#	Column	Meaning
1	chr_donorA	Donor (left segment) chromosome
2	brkpt_donorA	Donor breakpoint position
3	strand_donorA	Donor strand
4	chr_acceptorB	Acceptor (right segment) chromosome
5	brkpt_acceptorB	Acceptor breakpoint position
6	strand_acceptorB	Acceptor strand
7	junction_type	-1 (mates on either side of the junction) / 0 (non-canonical) / 1 (GT/AG) / 2 (CT/AC)
8	repeat_left_lenA	Length of repeat to the left
9	repeat_right_lenB	Length of repeat to the right
10	read_name	Source read name
11	start_alnA	Donor segment’s first aligned genomic position
12	cigar_alnA	Donor CIGAR; a paired segment carries the second mate after a p operation (the genomic gap between mates, negative when they overlap)
13	start_alnB	Acceptor segment’s first aligned genomic position
14	cigar_alnB	Acceptor CIGAR, same convention