SPLASH2 provides ultra-efficient, scalable, and unsupervised discovery on raw sequencing reads.

SPLASH is an unsupervised, reference-free, and unifying algorithm that discovers regulated sequence variation through statistical analysis of -mer composition, subsuming many application-specific methods. Here, we introduce SPLASH2, a fast, scalable implementation of SPLASH based on an efficient -mer counting approach. SPLASH2 enables rapid analysis of massive datasets from a wide range of sequencing technologies and biological contexts, delivering unparalleled scale and speed. The SPLASH2 algorithm unveils new biology (without tuning) in single-cell RNA-sequencing data from human muscle cells, as well as bulk RNA-seq from the entire Cancer Cell Line Encyclopedia (CCLE), including substantial unannotated alternative splicing in cancer transcriptome. The same untuned SPLASH2 algorithm recovers the BCR-ABL gene fusion, and detects circRNA sensitively and specifically, underscoring SPLASH2's unmatched precision and scalability across diverse RNA-seq detection tasks.