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methylpy add-methylation-level issue #53
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It is weird. Allc file should be sorted. I can look into this if you can share some files to reproduce the issue. Also, it would be helpful to check whether the bam file created is sorted. |
Thanks for the response. |
It will be great if you can provide the |
How about this? |
Thanks. Were you able to produce unsorted allc file from this bam file? Can you also point me to the genome (FASTA)? |
Were you able to produce unsorted allc file from this bam file? |
I don't think so. The allc file I got from the bam file is sorted. Here is the command I used
|
That is good to know. Here is what I used. Is there anything wrong that I did not realize with this? methylpy paired-end-pipeline And used this function add-methylation-level methylpy add-methylation-level Then I do can call DMR on the allc file properly. methylpy DMRfind |
The commands look good to me. Did you get unsorted allc files and end up with some error in the DMR finding step? |
Right. I got unsorted allc files. I could do DMR finding. But I got errors when I try to add methylation levels using the allc files. In the end, I just unzipped the allc file, sorted them, and then feed the files back to add methylation level, it worked. Anyway, as long as I did not get any errors or warnings with the DMR finding. That should be fine, correct? |
That is weird. I would recommend rerunning DMR finding on the sorted allc files to make sure things are working fine. |
I have a follow-up question- what does it mean if my output has NA? |
It means that there is no reads to estimate methylation level in that region in that sample. |
Hi
I guess my bam and allc file not sorted has to do with my settings of the number of processors and the sort memory being nonproportional to each other? |
Ah, that is good to know. You may want to drop You don't have to rerun from scratch. Manually doing samtools sort to get sorted bam file and then regenerating allc files using |
I have a similar issue to #48
But my output tsv file has only the bed file
This is the test bed file
chromosome start end
2 1 40001
2 40001 80001
2 80001 120001
2 120001 160001
This is the output I get
chromosome start end methylation_level_ACA methylation_level_ACB methylation_level_pCMT3-RNAiA methylation_level_pCMT3-RNAiB
2 1 40001
2 40001 80001
2 80001 120001
2 120001 160001
I checked that my files are using proper tab as delimiter and the allc files are not empty....
2 1647 - CAT 2 20 1
2 1649 + CGT 10 12 1
2 1650 - CGT 16 21 1
2 1653 + CCT 0 12 0
Here is the code
methylpy add-methylation-level
--input-tsv-file methylpy_wgenome.tsv
--output-file $outfile
--allc-files allc_ACA.tsv.gz allc_ACB.tsv.gz allc_pCMT3-RNAiA.tsv.gz allc_pCMT3-RNAiB.tsv.gz
--samples ACA ACB pCMT3-RNAiA pCMT3-RNAiB
--mc-type CHH
--num-procs 10
======
update- this is interesting. In the end I figured that it is because the allc files are not sorted. Shouldn't this be sorted automatically? This is my pair end pipeline code, did I miss anything???
methylpy paired-end-pipeline
--read1-files ./_1.fq.gz --read2-files ./_2.fq.gz --sample ${folderall[$indi]}
--merge-by-max-mapq True
--binom-test True
--unmethylated-control chloroplast
--min-cov 3
--forward-ref $fref --reverse-ref $rref --ref-fasta $reff
--num-procs 20 --sort-mem 35000000000
--path-to-output $directout
--remove-clonal True
--path-to-picard="/home/softwares/picard/"
--aligner-options "-p 8"
--trim-read False
1>$stdoutfile 2>$errfile
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