I am trying to run Bowtie2 alignment in a Nextflow pipeline, but I’m encountering an error where Bowtie2 can’t find the index files. The error message indicates that "reference_index.1.bt2" does not exist or is not a Bowtie 2 index.

I am trying to run Bowtie2 alignment in a Nextflow pipeline, but I’m encountering an error where Bowtie2 can’t find the index files. The error message indicates that "reference_index.1.bt2" does not exist or is not a Bowtie 2 index.

I have a simple pipeline:

I need to use REST API to download files in JSON. Then, I will run another process to update these files, for example, modify these files and submit these files to another database, etc. However, my code returns nothing. I learned from the first script and I am aware that Nextflow syntax looks like Groovy.

I have been using Nextflow for quite some time now, but today I am stuck with this error and I cannot really figure out why it is happening.

I am totally new to Nextflow.
I have some methylation files that I processed but ended up with a very low coverage.
I have 72 samples, and the facility gave us 72 fastaq files for run 1 read 1, 72 for run 1 read 2, 72 for run 2 read 1 and 72 for run 2 read 2. so basically I have 4 files per sample. They said “Sequenced across 6 lanes of 2 separate NovaSeq S4 2×150-bp runs (2 lanes in Run 1and 4 lanes in 2). Reads are concatenated across lanes by sample ID within each flow cell. You will need to concatenate reads by sample ID across the 2 flow cells to achieve the full depth for each sample”. Now, reading the Nextflow documentation i understood I could list all files in a single samplesheet.csv and Nextflow would recognize files from the same sample and concatenate them, so I ended up with a csv like this:

So my initial input file is an annotation file which contains the information for all the downstream process.
So this is my step1.nf

I’ve this structure:

So i have so far two nextflow script first one is parse_annotation.nf which generates a .csv file then that goes as input for sample_parse.nf. Now so far i have figured out from examples how to run as standalone workflow.