SALRR: Scalable Analysis of Long-Read RNA-Seq Enables Comprehensive Transcriptome Profiling in Human Brain

Isoform-resolved transcriptomics is fundamental to decoding the molecular complexity of the human brain, yet population-scale long-read RNA sequencing has remained inaccessible due to labor-intensive library preparation, sensitivity to RNA degradation in postmortem tissue, and the absence of integrated, reproducible analysis pipelines. Here we present SALRR (Scalable Analysis of Long-Read RNA-seq), an integrated wet-lab and computational platform designed to overcome these barriers. Automated ONT long-read cDNA library preparation on the Hamilton Microlab NGS STAR platform reduces hands-on time by 67% and enables 24 libraries per operator per day while maintaining performance across RNA integrity values. A modular, Snakemake-based pipeline performs end-to-end processing from ONT signal data to isoform-level quantification, incorporating SIRV spike-in calibration, multi-stage quality control, and stringent isoform validation. Applied to 10 postmortem frontal cortex samples from the North American Brain Expression Consortium, SALRR identified 31,607 high-confidence isoforms from 10,075 genes, including 8,532 novel splice variants absent from GENCODE v49, and complex splicing events systematically missed by short-read sequencing at neurodegeneration-relevant loci, including GBA1, CCNF, CHCHD10, and TREM2. All protocols and code are openly available, providing a scalable, community-ready framework for isoform-resolved transcriptomics in neurodegeneration, aging, and complex brain disease.