Problem about Methcounts

[keya...@gmail.com]

Hi，

I’m trying to build a pipeline about DNA methylation analysis, my mentor recommended  methpipe, which is really convenient, but I got some trouble when I ran “methcounts”, it came out all 0 in files, and I have changed some data ,but it still can’t work. Because of it, the following steps can’t work either.

chr1    3000000 -       CHH     0       0

chr1    3000005 +       CHH     0       0

chr1    3000010 +       CHH     0       0

chr1    3000014 -       CHH     0       0

chr1    3000020 -       CHH     0       0

chr1    3000029 -       CHH     0       0

chr1    3000034 +       CXG     0       0

chr1    3000036 -       CXG     0       0

……

chr7    3000262 +       CpG     0       0

chr7    3000263 -       CpG     0       0

……

Please let me know, what could be the possible reasons, which might be causing these issues. I'm a newbie to bioinformatics and don't have too much knowledge on DNA methylation, except some basic idea. So, any help will be appreciated.

 

All the best.

Ke Yang

The School of Life Science of Xiamen University

[Xiaojing Ji]

Hi Yang,

Can you print the first 10 lines of your input file (.mr file) for methcounts?

Best,

Liz

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Xiaojing Ji

Molecular and Computational Biology,

University of Southern California

1050 Childs Way, RRI 408c

Los Angeles, CA, 90089

[keya...@gmail.com]

Hi Liz ,

Thanks for reply, here is the first 10 lines of my input file (.mr.sorted_start). I followed the manual to use sorted file as input in methcounts.

chr1    2726187 2726236 SRX333391.11287706      6       -       AATATTAGGGTTGGTTTTTTTGTTTTTGTTTATGTTTTTTTGTTTTTGT       GGGGGGGGG9GGGGGGGGGGG,GGGBC8?CB/A@BGGGGGG/GGG@@5@

chr1    3000542 3000738 FRAG:SRX333391.12499511 0       +       TTATTTTTATAAAATTTGTTTTGTTTTTTAGCGGATTAAAATATTTAGGTTTTGTTGTTATAGGTTTTGGTGGTGTTATATTATGTTGGTTTTTTTTGATTATGTTTTTATATTGGAGGTTAGTAATTTAGGGTTAGATAATTATAGGTTTAGGTGTTGATTTTTGAGTTTTTGTTGGATGGGCGTTTTTGGTTTG    HHHHHHHHHHHHHHHHHGHHHHHHHHHHHHFGHHHHHGHFHHBHHHHHHCHHHHHHHFHHHHHFDHHDFHDHF@FHHHGHHHGHHHHHHHHHHHEGH@FHHGFGDE<FBGDGHGGG@GFBG@><>5GCHHHHHHEGHHHHHHEHGHHHHFHHHHHHHHHHHHEHHGHHEHHHHHHHHHHGHHHHDHHHHHHHGHHH

chr1    3000955 3001252 FRAG:SRX333391.7917718  1       -       GAAAATAAAAATATTAAGAATGTTTAAATAGTTATAAGAGATTATATTATTAATTTTTAGTATTGTTGAGTTTTTTTTTTTAGTTTTAGAGGATTTTAGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTTTTAGTATTTTTTTATTTATTTTATTATTTATTTTATTGTTGGGTTTTTATTTTTAATTTTGTTTGATTTTTTTGTTTAAGTATAGGTTATAATAGTTAG       GG=GGFGGDGEGGGD67,390'2;67::*<?:?@AGGGFEB<EE>GFGGCEGDFGGGGDE;FGFFFFBBGD?DDDEDFFFG@7GGGGFEBGB<G@EEBEABBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBFDDD8CBG@EGHHHHHHEHHHHGGHHHHHHHHHHEHHHHHHGHHEHGGGGGFCHHHFHHHGGGEGEHGHGHHHHDHFFFEFDHGHFHHHHHGFFGEFGFGG

chr1    3000980 3001081 SRX333391.9143093       0       -       TTTAAATTTAGTTTTTAAAGATTTATTTTAGTATTTTTTTATTTATTTTATTATTTATTTTATTGTTGGGTTTTTATTTTTAATTTTGTTTGATTTTTTTG   HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHFHHHHHHHHHHHHHHHHHHHHEGHHCHHHHHHHHFHHFHFHHHHHHHHHHGHHHHHHGHHHHEHHHHHH=

chr1    3001147 3001362 FRAG:SRX333391.11856730 0       -       TGGGATGGAAGTTGACGTGAGTAATAGAGAGGGGAATAGAAGAGTGTAAATTATTTAATTGAAAAAATGGAAAAAAAAAATTAGAATGATTTTAATTAGGGNNNNNNNNNNNNNATAAAAATATTAAGAATGTTTAAAAAGTTATAAGAGATTATATTATTAATTTTTAGTATTGTTGAGTTTTTTTTTTTAGTTTTAGAGGATTTTAGTTGTTT HHHHHHHHHHHHHHHHHHHEGEGGGGHHHFHHHHHHHHHHHHHHDGEGGFHHHHHGGHHHHHFHHHHHHHHHFFHHHG9GGFGEGGFGFGHHFFHHGDHFFBBBBBBBBBBBBB?'8@BBF-ABFAHBFH9DEHFBFFFF<BEBEFEEGF@?A5<CDD@;GCGEGBCC><866>9<@3:,>HHHEHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH

chr1    3002247 3002487 FRAG:SRX333391.1265244  1       +       ATGAAGTTTTTTATATAATTTATAATGTGAGAAATTATGTATTTTTGAATTTATGTTTTTAGAATTTTTTTATATAGTTTTAAGGGTTGTCGTGAAGATTANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGTGTTTTTTGGATTTATGATGTTTTTAATTATTATAGAGATATTGATTTTGGTAGGGAGAATATTGTTTGTTATAGGAATATAAAGTAAAGTAAATTATGT        HHHHHHFHHHHHHHHHHGHGHHHH>GGEGFHHGHHHHHHHHHHHHHHFFHHHGBHHHHHHHDHFHHFHHGHHGGEHGHHHGHHGHFFFHHGCCGFFHFH=@BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBEHHHHHHHHHHHHHHHFHHHGHHHHHHHHHHHHHFHHFHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHGHHHHHHHHHFHHHHHHHHHHH

chr1    3002301 3002550 FRAG:SRX333391.1376948  0       -       AGTAGGGTGTGGGTTTTTATGGTTGATAGTTGTTAGGTAGAAAGTTTGTTTGTATAATAATGTATATAATTTATTTTATTTTATGTTTTTGTGATAAATAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTGAGTTTAGGAGGTACGGGTGTATAGGTGTTTTTTAGTAAGGTGGTGAATATTGTGGTTTTTACGATAGTTTTTAAGATTGTATGAAAGAATTTTGAAAAT       HHHHHIGDGEIIIFGHHHHHHHHHHFHGHHHHHHHHHEHHEHHHEHHHHHHHHHHHHHHGHHFHHHHHHHHHHFHHHHHHHHHHHHHHHHHEGGGGFGEG?BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBHHHGHHHHHFEHHHHGHHHHGHGHHHHEHHHHHHHHHHFHHHFHHHHHGGHHHGHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH

chr1    3002539 3002742 FRAG:SRX333391.1288397  0       -       AGGTAATTAAAATGAAGTATAATAATAGATAAAGTTATTTTAATGAAATATGTGGAAAAAATAAGTGTTATATATAAGTATGTTAAATATAGTTTTAATTTNAATAGTAATAAATTAGAAAATATGTTGTTGAGTTTTATTATGTAGAATGGATGGTATGATATAGTTTTATTTTATTTTTGGATTAGAGTTAGTAGGGTGTG     HHHEHHHHHHHHHHHEHHHHHHGHGGHHGHHHHHEHHHHHHHHHHEHHHFHHHHHFHHGFHHEHHAGDFGHHHHHHGHDHGHHHHHHHHHHHEHHHHHGHEBHHEHHHHCDHHFGGHEBEEE8HHHHHHFHHHHHEHHHHHHHHFHHHHHHGGHHHHHHHHHHHHHHHHHHHHHHGGHHHHHHHHHHHHHHHHHHHHHHHHHH

chr1    3002539 3002742 FRAG:SRX333391.5030092  0       -       AGGTAATTAAAATGAAGTATAATAATAGATAAAGTTATTTTAATGAAATATGTGGAAAAAATAAGTGTTATATATAAGTATGTTAAATATAGTTTTAATTTNAATAGTAATAAATTAGAAAATATGTTGTTGAGTTTTATTATGTAGAATGGATGGTATGATATAGTTTTATTTTATTTTTGGATTAGAGTTAGTAGGGTGTG     HHHHHHHHHHHHGHGHHHHHHHHHGGHHHHHHHHHHHHHHHHHHHHHFHHHHFHFHHGHFEFHFGDHEHHFHHHHHHHFHHHFHHHGHGHGHFFFFGGGG;BFHFHHHGHEHGFHHHHEEEGDDDHFHHEHEHGGDGFDHGFHFHDFGGHHHHHFHGHHEEHGFDHFHGHHHHHHFHHHHHHHHHHHHHHHHHHHHHHHHHHH

chr1    3003006 3003336 FRAG:SRX333391.9646417  0       +       TTTAGTTTGAATTATTGTGAAATAAGTTAGTTTTTTTAGTTTATTAATGTAAAATTGTTATTTTTTTTAATTTTAAATTTTTTGTTTGTTTTTTTTTTTTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGATAATATGGTATTATATTTAAAAAATATTTTTTGTGTGTGTATAGGAGTTTTGTTTGATTGTATGTTTGTGTGTTTGGTGTTTTTAAGGTAAAAAGAGTG      HHHHHHHGHHGHHHGHHBGHHEEHEHFHHHFHGHHHHGGFHHHFEHHHHFHDDEGFHFDHEHHEHHHHHFGHEHAFDDG@FFFFFHEHHGHHHG7E=EEEEBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB9488>6BB,9CHFEHHEDBBE486<88<4<.EEE2EDHBHGGGGFFEE?C6DF>EEHHHHHFCHFFDDBFBHHHFFHHHHHHHHHHH@FHHHHHFHHHHHH

chr1    3003105 3003332 FRAG:SRX333391.3355405  0       +       TTTTGTTTAATTTTTTTTTAGAAAATTTATAAGTGATTTATTTTTTTGTATTTGGTTAGTGTTTATTGATTTAATTTATTTATATTATTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTATATTTAAAAAATATTTTTTGTGTGTGTATAGGAGTTTTGTTTGATTGTATGTTTGTGTGTTTGGTGTTTTTAAGGTAAAAAG     HHHHHHHHEBHFHFEHEH>?DFEE:?CCB<<CD9CB;DB?ECDD;D?<3%5=<5<4A=A5C>7C0=4%;@=<.:AECCC>=%2AAD<B4@BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBDAD9BFEDFECFC<BE@@ECHEHHFH@HHFGHGHEDED9FHEFHHH?HHHHHHHHHHEHFGHFHHHBHHFHHHHHBHEHHHFHH

All the best,

Ke Yang

在 2018年11月25日星期日 UTC+8下午5:20:13，Liz写道：

[Meng Zhou]

The methcounts file you looked at includes all cytosine sites, and for those you shared, they happened to be not covered by your reads.

Can you run levels on your methcounts file to get summary statistics? Then you can tell whether it's truely all zeros in your methcounts file, or it's just by chance that only the sites you have seen are not covered.

Also, I'm just curious, which mapper did you use? Your data were mapped to mm10, right?

Best regards,
Meng

[keya...@gmail.com]

Thank you, Meng and Liz,I checked the statistics files and  it seems that they are not all zeroes in my  methcounts file. I'm so sorry for my mistake. In this file, I mapped to mm9, but I also mapped other data to mm10.

By the way, I'm just curious that I coudn‘t use the paired-end test files ’snippet_1.fq‘ and ’snippet_2.fq‘, when I mapped them it shows that :

The number of reads in paired-end files should be the same.

I don’t know if I download the test files wrongly. 

Thanks again for your help!

All the best,

Ke Yang


在 2018年11月27日星期二 UTC+8上午7:22:28，Meng Zhou写道：

[Xiaojing Ji]

Hi Ke,

Have you been using the test reads data packaged in MethPipe? We had a check and found it to be outdated and unusable for our mapping program WALT. Please don't use this test data set and we'll replace it soon. You can still use WALT and MethPipe on other data. Thanks for reporting this and we apologize for the inconvenience.

Best,

Liz