Background Reference catalogs such as GENCODE capture most stably expressed mammalian genes but may not include condition-restricted or low-abundance transcripts. Reads supporting unannotated intergenic splice junctions are generally absent from annotation-restricted gene-count matrices; transcript assemblers may reconstruct a subset as novel models, but those models are typically handled separately from the annotated gene-count universe. TxNova is a lightweight command-line tool that directly indexes these unannotated splices from existing BAM files—without an external assembler, gffcompare, or workflow manager—yielding candidate leads for bench validation rather than standalone discovery claims.

Methods TxNova retains CIGAR N junctions from STAR/HISAT2 BAM files that recur in ≥2 samples and are absent from a comprehensive annotation, clusters them into residual loci, and counts each locus alongside annotated genes in a unified matrix. Structure gates—canonical splice motif, same-strand distance, coverage valley, bridging-junction absence, minimum length—remove likely artifacts to yield structure-pass models. An optional contrast filter retains loci detected in treatment but nearly silent in control.

Results A residual splice is a recurrent, unannotated CIGAR N junction that does not overlap any annotated gene body; intronic and antisense channels are out of scope. Across four published mouse treatment arms (GSE221720, GSE166522, GSE157460, GSE193335), harvest catalogs yielded 464, 657, 789, and 594 loci. On GSE221720, 45% of loci (209/464) shared an exact intron with another series, versus 0.074% for excluded junctions; a coordinate-placement control yielded 0/5,000 matches for length-matched intergenic decoys. Masked-gene recovery reached 87.6% (176/201) overall and 99.4% (176/177) among genes with a leak junction—sensitivity rather than precision estimates. An optional contrast provides a presence/absence screen on interval TPM. Residual models are partial reconstructions requiring cloning, RACE, or targeted proteomics for confirmation.

Conclusions TxNova produces a reproducible intergenic residual-locus catalog from existing BAM files, with four downloadable mouse injury/infection catalogs. Cross-series recurrence and coordinate-based null controls support reproducibility above simple placement backgrounds but do not by themselves establish biological validity. The optional contrast step offers a practical screen for candidate leads warranting experimental validation.