An error was reported in methylation extraction

[shoutong263]

Hello Felix

I encountered two errors after running step 3:

Sorting input file CpG_context_wwz-guo2_R1_bismark_bt2_pe.txt.chrChr01.methXtractor.temp by positions (using -S of 1500G) Died at /data01/masterHome/houtong/bismark/bismark2bedGraph line 487. Finished BedGraph conversion ...

and

No last chromosome was defined, something must have gone wrong while reading the data in (e.g. specified wrong file path for a gzipped coverage file?). Please check your command!

I tried a lot of things but I couldn't figure it out.

[shoutong263]

This is the command I used: nohup bismark_methylation_extractor -p --parallel 40 --comprehensive --no_overlap --bedGraph --cytosine_report --CX_context --split_by_chromosome --counts --buffer_size 1500G --report --samtools_path /data01/masterHome/bshoutong/samtools/samtools --genome_folder /data01/masterHome/bshoutong/jiajihua/Sch.index wwz-guo2_R1_bismark_bt2_pe.bam

[FelixKrueger]

OK let's try to dissect this:

Which type of genome are you using? is it chromosome or scaffold based? If the latter is the case, I would recommend you look at the option --scaffolds.

A rather minimal command could be:

bismark_methylation_extractor --bedGraph  wwz-guo2_R1_bismark_bt2_pe.bam

-p: this will be auto-detected. drop it --parallel 40: wow, that's a lot. How about starting with 4? --no_overlap: is the default for paired-end data. drop it --cytosine_report: can be added once the process is working --CX_context: this will make the process very slow. Are you working in a plant species? --counts: is the default. drop it --buffer_size 1500G: 1.5TB RAM? I doubt you will need more than a few GB --report: is the default. drop it

[shoutong263]

Thank you very much for your reply

I analyzed the plant genome, which has 14 major chromosomes and over 2,000 scaffolds at the level. I tried the --scaffolds parameter later, but came up with the same error. Also, the process file is really large, and I set it to 1500Gb to be on the safe side.

[FelixKrueger]

Hmm. Can you say until which step it worked? Did the methylation extraction work until completion? You should see the CpG, CHH and CHG context files, as well as the M-bias.txt file and a splitting report.

Has the bedGraph/coverage generation worked to completion? Do you see a .cov.gz file, and how many lines does it have? (gunzip -c - | wc -l). If this step has failed, you could start bismark2bedGraph using the C* context files as input. Something like:

bismark2bedGraph --gzip --CX -o outputfile.cov.gz --scaffolds C*

Or has it failed at the coverage2cytosine step?

Could to link the relevant text you see on screen when it is failing?