Residual barcode-like sequences and barcode-associated artifacts after Dorado and Barbell preprocessing of metagenomic reads

Barbell 0.3.2 applied to untrimmed Dorado basecalls reduced internal barcode-like matches to 0.0035–0.0037% while retaining 91.2–91.8% of input reads in blood mock-community data.

• Why it matters: Residual barcode-like sequences after ONT multiplex preprocessing can confound quality control, especially in low-biomass, host-dominated blood metagenomic samples.
• What they did: Blood datasets spiked with a microbial mock community and prepared with Native Barcoding Kit 96 V14 were processed with Dorado 1.1.1, Dorado 2.0.0, and Barbell 0.3.2, then screened for barcode-positive and internal barcode-like matches.
• The result: After demultiplexing, barcode-like sequences persisted in sample reads, and kit barcode sequences were detected in reference records (e.g., 66.7% of 96 barcodes with complete 24 nt identity), motivating careful preprocessing and downstream validation.

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