Barbell: Resolving Demultiplexing and Trimming Issues in Nanopore Data

Background Oxford Nanopore sequencing enables long-read sequencing across diverse applications, yet the experimental artifacts introduced by Nanopore barcoding are not well characterized. These artifacts can affect demultiplexing accuracy and downstream analyses. Results We performed a rapid barcoding experiment on 66 diagnostic samples and found that 83% of reads carried the expected single-barcode pattern, while 17% contained multiple barcodes or other artifacts. Current demultiplexers, including the widely used Dorado, fail to correctly handle these complex cases, leaving approximately 7% of reads partially trimmed and contaminated with adapter fragments. Additional issues include the presence of two barcodes at the same read endeither identical, originating from the same sample, or dierent, introduced after pooling. The latter can lead to barcode bleeding when the outer barcode is incorrectly selected. To address these problems, we developed Barbell, a pattern-aware demultiplexer that detects all barcode congurations. Barbell reduced trimming errors by three orders of magnitude, minimizes barcode bleeding, and its trimmed reads produce adapter-free assemblies. Conclusions Our results highlight the prevalence and impact of complex barcode attachments in Nanopore sequencing and demonstrate that Barbell drastically reduces their impact on downstream analyses. Barbell is open source and available at https://github.com/rickbeeloo/barbell