Hello, I hope you are doing well. I am trying to use Nextflow / nf-core to demultiplex a 10x cell-hashing (HTO) library. We have 10 hashing barcodes (15 bp) corresponding to 10 sample groups (mouse × condition × timepoint), and the hashing FASTQs are split across two lanes (L001/L002; paired-end). We validated the barcode is in R2 (starts at position 11), so we used R2 read structure 10S15B65T and ignored R1 (28S). We ran nf-core/demultiplex with --demultiplexer fqtk (v1.7.1, docker). Running both lanes together repeatedly failed with an error like “observed_bases 30 vs expected_bases 15”. Running lane-by-lane with per-lane tar.gz inputs worked for both lanes, and we combined the outputs by concatenating the per-sample *.fastp.fastq.gz files across lanes. Could you help confirm: 1. Is lane-by-lane the recommended/expected approach for fqtk with multi-lane inputs, or is there a correct way to run both lanes together? 2. Is concatenating demultiplexed FASTQs across lanes the right way to combine outputs? 3. Does this pipeline provide (or is there a recommended way to generate) a cell-barcode → sample assignment table for downstream integration? Our end goal is to label cells for GEX/VDJ analyses and ultimately build Dowser lineage trees with nodes labeled by GEX-derived cell type. Thanks a lot for any guidance you can share!