Barbell reveals and resolves demultiplexing and trimming issues in Nanopore data.

motivationOxford Nanopore sequencing enables long-read analysis for diverse applications, but artefacts introduced by Nanopore barcoding are poorly characterized and can compromise demultiplexing accuracy and downstream analyses.

resultsUsing a rapid barcoding experiment on 66 diagnostic samples, we found that only 83% of reads followed the expected single-barcode configuration, while 17% showed complex barcode attachments. We observed similar patterns in public datasets, and also in native barcoding datasets where only 30%-70% of the reads had barcodes on both ends. Widely used demultiplexers, including Dorado, fail to resolve these cases, leaving ∼10% of our rapid barcoding reads partially trimmed and contaminated with adapter fragments. We developed Barbell, a pattern-aware demultiplexer that is designed to detect complex barcode configurations. Barbell reduced contaminated reads from >400 000 (Dorado/Flexiplex) to 166 (99.96% reduction), minimized barcode bleeding, and supports custom experimental designs such as dual-end barcodes and shorter barcodes (e.g. Illumina barcodes). We further show that such contamination is widespread in public databases, with Nanopore sequences detected in hundreds of NCBI entries, some of which are responsible for artificial taxonomic connections. AVAILABILITY AND IMPLEMENTATION: Barbell is open source and available at https://github.com/rickbeeloo/barbell.