Arnaiz del Pozo, C.; Sanchis-Lopez, C.; Huerta-Cepas, J.

The combination of target capture metagenomics and long-read sequencing represents a powerful approach for the characterisation of rare microbial taxa and their functional genes. However, standard Nanopore library preparations are incompatible with established capture protocols. A possible workaround is the preparation of Illumina libraries prior to ONT sequencing. Currently, this hybrid approach is hindered by a lack of specialised demultiplexing software capable of handling residual adapter fragments; Nanopore's higher error rates and positional variability. Here, we present deluxpore: a Nextflow pipeline that demultiplexes Nanopore reads from Illumina dual-indexed libraries (NEBNext and Nextera) using BLAST alignment and Levenshtein distance matching. Extensive benchmarking across 18 replicates validates the viability and precision of this hybrid indexing approach. Benchmarking demonstrates that accurate demultiplexing requires minimum Q20 data quality and strategic index selection. Unique index-to-sample designs achieved 91.7% sample recovery at Q20 versus 46.1% for combinatorial approaches. We also identified high-crosstalk index pairs within NEBNext Primer Set A and provide an optimized 8-sample configuration achieving ~95% accuracy at Q20. deluxpore enables reliable, automated demultiplexing for hybrid capture-long-read sequencing workflows.