diff --git a/CHANGELOG.md b/CHANGELOG.md index b6e4893..91d4370 100644 --- a/CHANGELOG.md +++ b/CHANGELOG.md @@ -99,6 +99,12 @@ Sections commonly used: Features, Bug fixes, Other changes. ### Bug fixes +- `--soloUMIfiltering MultiGeneUMI_CR` kept every gene tied at the + highest read count; CellRanger gives a tied UMI to no gene at all. + Since one read per gene is the ordinary shape of a multi-gene UMI, the + flag removed nothing in practice. On a 20k-read 10x fixture the count + matrix moves from 16 465 to 15 414 against STAR's 15 423. + - **STARsolo `Gene` assignment now requires exon concordance**, matching STARsolo: a read counts toward a gene only when every aligned block lies within the gene's exons, rather than merely overlapping one. This diff --git a/src/solo/count.rs b/src/solo/count.rs index 9af3edf..7d906c8 100644 --- a/src/solo/count.rs +++ b/src/solo/count.rs @@ -822,8 +822,43 @@ fn filter_multi_gene_umi(genes: &HashMap, filtering: UmiFiltering) -> let thresh = if max == 1 { 2 } else { max }; genes.iter().filter(|&(_, &rc)| rc >= thresh).collect() } - // CellRanger > 3.0: keep the highest-read-count gene(s); no singleton drop. - UmiFiltering::MultiGeneUmiCr => genes.iter().filter(|&(_, &rc)| rc >= max).collect(), + // CellRanger: the gene with the strictly highest read count takes the + // UMI, and a tie gives it to nobody. + // + // STAR `SoloFeature_collapseUMIall.cpp:212-224` walks the genes keeping + // a running maximum, and clears its winner whenever it meets an equal + // count: + // + // ```cpp + // if (ig.second>maxu) { maxu=ig.second; maxg=ig.first; } + // else if (ig.second==maxu) { maxg=-1; }; + // ... + // if ( maxg+1==0 ) continue; // not counted for any gene + // ``` + // + // The outcome does not depend on the order the genes are visited: a + // strict maximum always ends as the winner, and any tie at the maximum + // always ends with none. So iterating a `HashMap` here is safe. + // + // This previously kept every gene tied at the maximum, which is the + // opposite decision on exactly the case the rule exists for, and made + // the flag inert on the common shape of one read per gene. + UmiFiltering::MultiGeneUmiCr => { + let mut best_count = 0u32; + let mut winner: Option<&u32> = None; + for (gene, &rc) in genes { + if rc > best_count { + best_count = rc; + winner = Some(gene); + } else if rc == best_count { + winner = None; + } + } + match winner { + Some(gene) => vec![(gene, genes.get(gene).expect("winner is a key"))], + None => Vec::new(), + } + } UmiFiltering::None => unreachable!(), } } @@ -2057,6 +2092,43 @@ mod tests { assert!("bogus".parse::().is_err()); } + /// The case the rule exists for, and the one the old code got backwards: + /// when two genes tie on read count, CellRanger counts the UMI for + /// neither. STAR clears its winner on an equal count + /// (`SoloFeature_collapseUMIall.cpp:212-224`) and skips the UMI when no + /// strict maximum survives. + /// + /// One read per gene is the common shape of a multi-gene UMI, so keeping + /// the ties made `--soloUMIfiltering MultiGeneUMI_CR` inert in practice: + /// on a 20 000-read 10x fixture it removed nothing at all, against 1 030 + /// counts removed by STAR. + #[test] + fn multi_gene_umi_cr_drops_a_tie_entirely() { + let mut tied = HashMap::default(); + tied.insert(0u32, 1u32); + tied.insert(1u32, 1u32); + assert!(filter_multi_gene_umi(&tied, UmiFiltering::MultiGeneUmiCr).is_empty()); + + // A tie at the maximum loses even when a third gene sits below it. + let mut tied_with_loser = HashMap::default(); + tied_with_loser.insert(0u32, 5u32); + tied_with_loser.insert(1u32, 5u32); + tied_with_loser.insert(2u32, 3u32); + assert!( + filter_multi_gene_umi(&tied_with_loser, UmiFiltering::MultiGeneUmiCr).is_empty(), + "a tie at the maximum takes the UMI from everyone, including the third gene" + ); + + // A strict maximum still wins, whatever else is present. + let mut strict = HashMap::default(); + strict.insert(0u32, 5u32); + strict.insert(1u32, 4u32); + strict.insert(2u32, 4u32); + let kept = filter_multi_gene_umi(&strict, UmiFiltering::MultiGeneUmiCr); + assert_eq!(kept.len(), 1); + assert_eq!(*kept[0].0, 0); + } + #[test] fn multi_gene_umi_cr_keeps_top_gene() { // UMI maps to gene 0 (3 reads) and gene 1 (1 read). CR keeps only gene 0.