Hello, I hope you are doing well. I am a graduate research assistant at the Columbia University Herbert and Florence Irving Institute of Cancer Dynamics. I need some support using nextflow to do the following tasks: • Use nextflow to demultiplex for 10 samples from antibody hashing barcodes. There are 4 fastq files, (two pairs of R1 and R2 data with the 10 samples spread across both pairs of files) • Run the gene expression pipeline on the demultiplexed data • Run airrflow on the data to generate b-cell repertoires I am struggling with the first part as it seems like nextflow demultiplexer expects 10 pairs of fastq files (please see the error below). I have attached my supporting csv files and the nextflow command that I tried to run. Can you please provide feedback on my approach? Thank you, Christine Sharabun

Your image suggests you are passing samplesheet.csv to demultiplex, but you want to pass the csv file you have called run_info

Indeed

Thank you! I readjusted my nextflow command to: nextflow run nf-core/demultiplex \ --demultiplexer fqtk --input /home/christine/iicd/rseq_bcells/.nextflow/run_info.csv \ --outdir /home/christine/iicd/rseq_bcells/.nextflow/results \ -profile docker

I don't know how fqtk works, but I do know it is currently broken on AWS: https://github.com/nf-core/demultiplex/issues/338 The internal pipeline plumbing for fqtk is set up in a very unusual way (hence it doesn't work on AWS) Can you run ls on that work directory it has given in the error?

Hello, when ls run ls on that directory, it confirms there are no fastq files in that directory.

what is in that directory?