Rescuing the "Unclassified": An Automated Pipeline for Maximizing Sequence Yield in Multiplexed Nanopore 16S rRNA Sequencing

Multiplexed 16S rRNA gene sequencing using Oxford Nanopore Technologies frequently results in a significant proportion of reads being categorized as "unclassified" by the standard Dorado demultiplexer. We present a fully automated Bash-based pipeline designed to recover and accurately re-attribute these unclassified reads to their corresponding barcode. By implementing iterative sequence-based alignment via BLASTn and size-filtering constraints, our tool improves data utilization and taxonomic depth. Preliminary testing demonstrates a robust recovery of previously discarded sequences, providing a cost-effective solution for improving the resolution of microbiome studies.