Isoform-resolved transcriptomics is essential to understanding the molecular complexity of the human brain, yet population-scale long-read RNA sequencing has been limited by several practical and analytical challenges. The authors present SALRR (Scalable Analysis of Long-Read RNA-seq), an integrated platform combining an automated wet-lab workflow with a reproducible computational pipeline intended to make isoform-level long-read transcriptomics accessible for studies of aging, neurodegeneration, and complex brain disease. The platform was designed specifically to address three common barriers: labor-intensive library preparation, sensitivity of postmortem RNA to degradation, and the prior absence of an integrated, reproducible analysis pipeline.
SALRR's wet-lab component automates Oxford Nanopore Technologies (ONT) long-read cDNA library preparation using the Hamilton Microlab NGS STAR platform. Automation reduced operator hands-on time by 67% and enabled preparation of up to 24 libraries per operator per day. The automated protocol maintained performance across a range of RNA integrity values, which is important for postmortem brain tissue that can exhibit variable RNA quality. The authors report that this automated approach makes library preparation less labor-intensive and more scalable for population-level studies.
The computational arm of SALRR is a modular, Snakemake-based pipeline that performs end-to-end processing from ONT signal-level data to isoform-level quantification. The pipeline incorporates SIRV spike-in calibration to support quantitative interpretation, multi-stage quality control steps, and stringent isoform validation criteria. This integrated workflow is intended to produce reproducible, validated isoform calls and standardized outputs suitable for downstream analysis and comparison across samples. The modular design allows components to be adapted or substituted while preserving an end-to-end, reproducible framework.
The authors applied SALRR to 10 postmortem frontal cortex samples sourced from the North American Brain Expression Consortium. Using the integrated wet-lab and computational pipeline, SALRR identified 31,607 high-confidence isoforms originating from 10,075 genes. These results demonstrate the platform's capacity to generate deep isoform-resolved transcriptome profiles from human brain tissue, even when starting RNA quality varies, and highlight the potential for population-scale application.
Among the high-confidence isoforms discovered, 8,532 represented novel splice variants that were absent from GENCODE v49. The identification of thousands of novel isoforms indicates that long-read sequencing through SALRR can reveal substantial unannotated transcript diversity in human brain tissue. The study emphasizes that these novel isoforms were validated within the pipeline's stringent criteria, although specific validation metrics and thresholds are reported in the source and should be consulted directly for technical details.
SALRR uncovered complex splicing events at genes implicated in neurodegenerative disease, including GBA1, CCNF, CHCHD10, and TREM2. The authors note that these complex events were systematically missed by short-read sequencing approaches, underscoring the advantage of long-read, isoform-resolved data for resolving transcript complexity at loci of clinical and biological interest. The platform therefore has potential to refine transcript models and to inform studies into mechanisms of neurodegeneration where alternative splicing or unannotated isoforms may be relevant.
All laboratory protocols and computational code for SALRR are made openly available by the authors. By distributing both the automated wet-lab protocols and the Snakemake pipeline, the study aims to provide a community-ready framework that other investigators can adopt or adapt for isoform-resolved studies of aging, neurodegeneration, and complex brain disease. The authors declare no competing interests, list NIH and ZIA funding sources, and released the preprint under a CC0 license.
SALRR addresses key barriers that previously limited population-scale long-read RNA-seq: the labor and time required for library preparation, sensitivity to RNA degradation in postmortem tissue, and the lack of integrated, reproducible analysis pipelines. The automated ONT library preparation and SIRV-calibrated Snakemake pipeline collectively deliver a scalable workflow that produced over 31,000 validated isoforms from ten human frontal cortex samples, including thousands of novel splice variants. Specific methodological parameters, validation thresholds, and detailed performance metrics are provided in the source material and supplementary files; readers should consult those materials for implementation and technical replication.
SALRR provides an integrated, scalable solution for isoform-resolved long-read RNA sequencing in human brain, coupling automated ONT library preparation with a reproducible computational pipeline. The platform successfully identified tens of thousands of high-confidence isoforms, including thousands absent from GENCODE v49, and resolved complex splicing events at neurodegeneration-relevant loci that were not captured by short-read methods. Open access to protocols and code positions SALRR as a community-ready resource for advancing transcriptomic studies in neurodegeneration, aging, and complex brain disorders.