Tranquillyzer: A Flexible Neural Network Framework for Structural Annotation and Demultiplexing of Long-Read...

Long-read single-cell RNA sequencing using platforms such as Oxford Nanopore Technologies (ONT) enables full-length transcriptome profiling at single-cell resolution. However, high sequencing error rates, diverse library architectures, and increasing dataset scale introduce major challenges for accurately identifying cell barcodes (CBCs) and unique molecular identifiers (UMIs) - key prerequisites for reliable demultiplexing and deduplication, respectively. Existing pipelines rely on hard-coded heuristics or local transition rules that cannot fully capture this broader structural context and often fail to robustly interpret reads with indel-induced shifts, truncated segments, or non-canonical element ordering. We introduce Tranquillyzer (TRANscript QUantification In Long reads-anaLYZER), a flexible, architecture-aware deep learning framework for processing long-read single-cell RNA-seq data. Tranquillyzer employs a hybrid neural network architecture and a global, context-aware design, and enables precise identification of structural elements - even when elements are shifted, partially degraded, or repeated due to sequencing noise or library construction variability. In addition to supporting established single-cell protocols, Tranquillyzer accommodates custom library formats through rapid, one-time model training on user-defined label schemas, typically completed within a few hours on standard GPUs. Additional features such as scalability across large datasets and comprehensive visualization capabilities further position Tranquillyzer as a flexible and scalable framework solution for processing long-read single-cell transcriptomic datasets.