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
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