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[PalmaGudiel]

Hi,

I found your MethylStar paper and pipeline while searching for straightforward methods for WGBS data preprocessing and I was particularly drawn to it since it is specifically mentioned that it is suitable for both experts and non-experts (I fall in the latter category). I am very used to work with R programming, but I am completely new to the ubuntu/bash environment so my question might be rather dumb!

While changing the configuration to set the proper paths to each requested file, I noticed that MethylStar is not recognizing my .fastq files, any idea why would that be? After specifying the corresponding path in the first step of the configuration "1. Path: RAW files" I get the following messages:

Founded 0 files in the directory.

Also, the size of your data-set almost: 4.8G

So, my 920 .fastq files do take up almost 4.8G but MethylStar does not seem to be recognizing the individual files as suitable input raw files.

I have tried running the first step of the pipeline (trimmomatic) but I get the following error messages: _Starting Trimmomatic ... Trimmomatic finished. Total time 0 Minutes. sort: cannot read: /home/palmagudiel/export_results/trimmomatic-files/list-finished.lst: No such file or directory rm: cannot remove '/home/palmagudiel/exportresults/trimmomatic-files/list-finished.lst': No such file or directory Something went wrong... something is going wrong... please run again.

But I don't really know if that's related to the "founded 0 files" issue or if it's completely unrelated.

Thank you very much in advance!!

[shahryary]

Hi @PalmaGudiel
Thank you for using our pipeline; As I understand, the problem is in the path directory of raw files; in other words, you can't pass just the file path to the pipeline; you should create a folder for your raw files and then give that folder path to the pipeline. For instance, create a folder in /home/palmagudiel/rawFiles/ and copy all the fasta files into there; you should pass /home/palmagudiel/rawFiles/ to the pipeline.

Also, there is a video tutorial that might help to configure the pipeline.

Please let me know if you have any questions.

[PalmaGudiel]

Hi @shahryary Thank your for your fast reply. I had already given the folder path to the pipeline (please see caption below).

I have also been checking the video tutorial for every step of the way, it's been useful to watch for other issues, just not for this particular one.

[shahryary]

@PalmaGudiel thanks for your reply; Could you please send me: 1- all the configuration parameters (from main menu --> 'C' --> number 10 "see configured parameters"). 2- in linux bash please do ls -lah and send just head of lists. Thanks.

[PalmaGudiel]

Yes, of course!

Please keep in mind that I am still at the beginning of the pipeline, I am aware that there might be several unresolved issues other than the problem with the raw files location/identification.

1) Configuration parameters:

Here is a summary of my current configuration parameters (it's a work in progress, right now I am focusing on being able to run at least the trimmomatic part):

• RAW files location: /home/palmagudiel/fastq/
• Number and Size of the data-set: 0 Files and Total size: 4.8 Gigabyte
• The directory of results: /home/palmagudiel/export_results
• Genome type: Human
• Genome folder location: /home/palmagudiel/MethylStar -- Genome Reference name: ((I am still trying to figure out where to obtain this file))
• Paired End: Disabled
• Trimmomatic location: /usr/share/trimmomatic/ -- JAVA path: /usr/lib/jvm/java-16-openjdk-amd64/bin/java -- ILLUMINACLIP: /usr/share/trimmomatic//adapters/TruSeq3-PE.fa:1:30:9 -- LEADING: 20 -- TRAILING: 20 -- SLIDINGWINDOW: 4:20 -- MINLEN: 36 -- Number of Threads: 8
• QC-Fastq path: software/FastQC/fastqc
• Bismark parameters: /software/Bismark -- scBS-Seq (--pbat)? Disabled -- Nucleotide status: false -- Number of Parallel: 8 Threads. -- Buffer size: 40 Gigabyte. -- Samtools Path: /bin/samtools -- Bedtools Path: /usr/bin/bedtools -- Intermediate for MethExtractor: Disabled
• Methylation extraction parameters( Only for quick run) -- Minimum read coverage: 1
• Methimpute Part: -- Methimpute Intermediate : Enabled -- Methimpute probability(Intermediate): constrained -- Methimpute Fit reports: Enabled -- Methimpute Enrichment plots: Enabled -- Methimpute Full report: Enabled -- Methimpute Context: All
• Parallel mode is: Disabled
• E-mail notification: Disabled
• MethylStar version: 1.1.1

2) ls -lah (you meant in the fastq folder with the raw files, right?)

Thanks again!

[shahryary]

@PalmaGudiel thanks for sharing the configs. I see the problem; the Pipeline reads all the files in zip format (usually trimmomatic use this format to improve performance), so all your files should be converted into fastq.gz format. It's easy; you can do it with linux: go inside of the raw folder and run: gzip *.fastq. It should zip all the files inside of the folder. Now, you can re-config the path.

Also, I see you didn't configure the QC-Fastq path if you need the report, you should configure this option too.

[PalmaGudiel]

I knew for sure it was something dumb like this, sorry to have bothered you so much! It runs now, thank you very much!