CellRanger4 + clip5pNbases/clip3pNbases misplaces reads by clip5pNbases

[Psy-Fer]

Summary

--clipAdapterType CellRanger4 combined with a non-zero --clip5pNbases / --clip3pNbases misplaces most reads by exactly clip5pNbases. Each option on its own is fine; only the pairing breaks.

Found while validating the CR4 clip work in the hyalite PR (#198 ). It predates that PR and is not caused by it.

Reproduction

Real 10x mouse chr19 (see CONTRIBUTING.md for dataset) , rustar and STAR 2.7.11b with the same flags:

SC=test/data/singlecell
COMMON="--readFilesIn $SC/reads/S10_L001_R2_001.fastq.gz $SC/reads/S10_L001_R1_001.fastq.gz \
  --readFilesCommand zcat --soloType CB_UMI_Simple \
  --soloCBwhitelist $SC/reference/10xv2_whitelist_sub50K.txt --soloFeatures Gene \
  --clipAdapterType CellRanger4 --clip5pNbases 5 --clip3pNbases 3 \
  --outSAMattributes NH HI AS nM --outSAMtype SAM --runThreadN 4"

./target/release/rustar-aligner --runMode alignReads --genomeDir $SC/solo_idx \
  --sjdbGTFfile $SC/reference/gencode.vM19.annotation.chr19.gtf \
  $COMMON --outFileNamePrefix $SC/rustar_cr4clip_

STAR --genomeDir $SC/star_solo_idx $COMMON --outFileNamePrefix $SC/star_cr4clip_

Then histogram the per-read POS delta (rustar − STAR) over primary records present in both.

What it looks like

POS delta, ~16.8k reads in both:

delta	reads
−5	8527
+5	3546
0	552

−5 and +5 are exactly clip5pNbases. Same read at the same POS carries CIGARs differing by 5 in the leading soft clip, e.g. 5S82M3S (rustar) vs 10S77M3S (STAR).

Mapped counts stay close — 14747 vs 14687 uniquely mapped — so reads are being misplaced, not lost, which is why Log.final.out looks healthy.

Scope: only the combination

configuration	result
CellRanger4 alone	fine — 0 clip diffs, 900 other (the usual mapping/tie baseline)
--clip5pNbases 5 --clip3pNbases 3 alone (no CR4)	fine — 1 leading-clip diff, 980 other
both together	broken — 15605 differing, dominated by ±5

Notes for whoever picks this up

• Not apply_read_clips (src/io/sam.rs:1106). It already swaps lead/trail for reverse strand and does not touch POS. I checked this first; it's clean. The cause is upstream, in what sequence reaches the aligner or how POS is derived from it.
• The bidirectional ±5 split (~70/30) suggests something strand-dependent, but that is a hypothesis, not a finding.
• The POS-delta histogram is the diagnostic that made this obvious. CIGAR-level diffing hid it, because the CIGARs look individually plausible.

Dead end — please don't repeat it

STAR's ClipMate::clip does the fixed N-trim before the adapter trim, so our poly-A scan window is formally wrong for this pairing (we scan raw[TSO..], STAR scans raw[TSO+clip5p .. len-clip3p]). I tried narrowing the window to match, and agreement got worse: leading-clip diffs went 475 → 549, because the position bug swamps the window effect. It's reverted, with an NB comment at solo::clip_adapter_cr4_with_tso.

Fix the position bug first; then the window correction can be measured honestly.

Why it matters now

#176 makes clip_adapter_type = "CellRanger4" a default on 10x geometry (CELLRANGER_DEFAULTS). Today CR4 is opt-in, so this only bites users who asked for it. If #176 lands first, any 10x user who adds --clip5pNbases hits this without ever typing --clipAdapterType CellRanger4.

This should be fixed before #176 merges.

[Psy-Fer]

Details above are the formal breakdown of the bug.

This will get picked up as I move through the various PR's, but if someone is bored and wants to try fixing it before I get there, that's fine too.

Cheers,
James