# Help running Nextflow methylseq on multiple fastq files per sample

**URL:** <https://community.seqera.io/t/help-running-nextflow-methylseq-on-multiple-fastq-files-per-sample/859>\
**Category:** Ask for help\
**Created:** [June 18, 2024, 12:14am UTC](https://community.seqera.io/t/help-running-nextflow-methylseq-on-multiple-fastq-files-per-sample/859 "2024-06-18T00:14:41Z")\
**Posts on this page:** 1\
**Page:** 1

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**Author:** ![coldkiss](https://dub1.discourse-cdn.com/flex013/user_avatar/community.seqera.io/coldkiss/32/772_2.png) [@coldkiss](https://community.seqera.io/u/coldkiss)\
**Post date:** [June 18, 2024, 12:14am UTC](https://community.seqera.io/t/help-running-nextflow-methylseq-on-multiple-fastq-files-per-sample/859/1 "2024-06-18T00:14:41Z")

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Hello, 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 2x150-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:

> sample, fastq1, fastq2  
> X1101560932,X1101560932\_S1\_L001\_R1\_001.fastq.gz,X1101560932\_S1\_L001\_R2\_001.fastq.gz  
> X1101560932,X1101560932\_S1\_R1\_001.fastq.gz,X1101560932\_S1\_R2\_001.fastq.gz

I run the Nextflow methylseq pipeline like this:

> nextflow run nf-core/methylseq -profile conda -c nextflow.config --input samplesheet.csv --genome GRCh38 --single\_end false --em\_seq --outdir home/wbc -resume -bg

And everything worked, I ended up with a single coverage file for each sample, but with extremely low coverage. I asked a person with expertise in these kind of data and she said sequencing library etc is allright from the QC reports, and she suggested _“merge fastq or BAMs (before deduplication) across the two flow cells (per samples) to get an overview of mean coverage in the two combined runs. I think the coverage reported now is only based on the first flow cell”_

Now, if I concatenate files with cat

```auto
cat "$run1_r1_file" "$run2_r1_file" > "${sample_id}_R1_combined.fastq.gz"

```

and rerun the pipeline it gives errors on fastqc and exit the pipeline, what am I supposed to do? in the bismark folder of my Nextflow run on non-concatenated files i had the folders: _alignments deduplicated methylation\_calls preseq reports summary_ but bam files are just in deduplicated folder, so I don’t understand what to do please help