Mapping Bisulphite converted sequence

[kalyankpy]

Hi Yuanxin,

I am quite interested in BSMAP. I am working on RRBS data from stem cells. I am trying to install BSMAP on my computer (Ubuntu 11.10, 64 bit). But it stops with an error. I posted the error messge at http://code.google.com/p/bsmap/issues/detail?id=7 . Can you fix this!

Kalyan

[yxi]

Hi Kalyan,

The error is in compiling samtools, bsmap used samtools API to read SAM files.

After google search I found someone experienced similar problems.

http://bugs.debian.org/cgi-bin/bugreport.cgi?bug=606004

Hope this could help

Thanks,

Yuanxin

Originally Posted by kalyankpy

Hi Yuanxin,

I am quite interested in BSMAP. I am working on RRBS data from stem cells. I am trying to install BSMAP on my computer (Ubuntu 11.10, 64 bit). But it stops with an error. I posted the error messge at http://code.google.com/p/bsmap/issues/detail?id=7 . Can you fix this!

Kalyan

[kalyankpy]

Hi Yuanxin,

I went through the link suggested by you! I noticed that there was a bug in the samtools in earlier versions which was rectified after 0.1.13 version (I noticed that the samtools you have provided with bsmap is 0.1.7).
I tried to overcome this bug by trying to download and install samtools-0.1.17 version from ubuntu repositories. I could install it successfully from repository. I also tried by downloading the sourcecode and compiled it with 'make' command. It workd perfectly. Assuming that problem is in the samtools, I replaced the samtools folder in bsmap with the newer version of samtools source code (0.1.18) and then tried compiling (with 'make').

This option took me few step ahead. Here I am attaching the output of the 'make' command. For me the problem doesnt seem to be in samtools rather it seems to be in some code that links BSMAP and SAMTOOLS (I am not a good programmer, I am learning! Its only my assumption. As a programmer you can analyze better!).

[yxi]

Good to know you solved it. We experienced a runtime error using the later versions of SAMTOOLS on Power7 CPU based system. So BSMAP included an early version SAMTOOLS.

Originally Posted by kalyankpy

Hi Yuanxin,

I went through the link suggested by you! I noticed that there was a bug in the samtools in earlier versions which was rectified after 0.1.13 version (I noticed that the samtools you have provided with bsmap is 0.1.7).
I tried to overcome this bug by trying to download and install samtools-0.1.17 version from ubuntu repositories. I could install it successfully from repository. I also tried by downloading the sourcecode and compiled it with 'make' command. It workd perfectly. Assuming that problem is in the samtools, I replaced the samtools folder in bsmap with the newer version of samtools source code (0.1.18) and then tried compiling (with 'make').

This option took me few step ahead. Here I am attaching the output of the 'make' command. For me the problem doesnt seem to be in samtools rather it seems to be in some code that links BSMAP and SAMTOOLS (I am not a good programmer, I am learning! Its only my assumption. As a programmer you can analyze better!).

[kalyankpy]

Hi, The problem still xists. I dont know if my message is clear for you. I could successfully install SAMTOOLS as a standalone application. However, I fail to install it when it is part of bsmap. Even if I replace the older version of SAMTOOLS with the newer version inside the BSMAP folder, the problem continues.

I liked the logic of your programme. I am just waiting for a solution to this installation problem.

Is it possible for you to send me the executibles for a 64 bit ubuntu system that can work for me (so that I dont need to compile them on mine as this problem persists)

[kalyankpy]

Hi,

Yes finally the problem is solved. Here I explain the steps that help me resolve the installation steps in Ubuntu 11.04

tweak the makefile in the bsmap directory as follows

original line "$(CC) $(FLAGS) $(LIBS) $(REF_MODE) $(OLIGOLEN) $^ -o $@ $(THREAD) -lz -lbam"

Change it as "$(CC) $(FLAGS) $(LIBS) $(REF_MODE) $(OLIGOLEN) $^ -o $@ $(THREAD) -lbam -lz"

replace the older version of samtools source directory with the new version - 0.1.17

I dont understand why changing the order of "-lz -lbam" to "-lbam -lz" solves this issue.

[aniruddha.otago]

Hi Guys,

Recently we have published a paper in Nucleic Acids research- "Comparison of alignment software for genome-wide bisulphite sequence data". you might find it useful to have a look at it.. http://www.ncbi.nlm.nih.gov/pubmed/22344695

[rskr]

Originally Posted by aniruddha.otago

Hi Guys,

Recently we have published a paper in Nucleic Acids research- "Comparison of alignment software for genome-wide bisulphite sequence data". you might find it useful to have a look at it.. http://www.ncbi.nlm.nih.gov/pubmed/22344695

Doesn't look like you compared the accuracy of the mapping algorithms say by generating randomized cpg data. Any good reason why this wasn't done?

[yxibcm]

The BSMAP version (v1.02/v1.2) used in this paper is a bit old. You may want to use the latest version (v2.5) to get an up-to-date comparison.

Originally Posted by aniruddha.otago

Hi Guys,

Recently we have published a paper in Nucleic Acids research- "Comparison of alignment software for genome-wide bisulphite sequence data". you might find it useful to have a look at it.. http://www.ncbi.nlm.nih.gov/pubmed/22344695

[rskr]

Originally Posted by yxibcm

The BSMAP version (v1.02/v1.2) used in this paper is a bit old. You may want to use the latest version (v2.5) to get an up-to-date comparison.

I guess old funky data "The accuracy went up when they trimmed it", has to get published somehow.

[aniruddha.otago]

yeah when we were performing the analysis the latest version was 1.2 ( release, April, 2011). every program has newer version each week, this analysis was done at that time with latest versions of all the aligners at that particular time.

Cheers,

Aniruddha.

[aniruddha.otago]

Hi Yuanxin,

Thanks, we have solved the problem and it came up with reasonable percentage of methylation. It was useful for the analysis. Thanks.

[aniruddha.otago]

Hi rskr,

We never really thought of trying to do that. We were attempting
to see the performance with real RRBS data which we did. I'm not
sure that going to the trouble of trying to generate random CpG fragments
would have achieved very much at all. Any attempt to 'randomise' the
C/T conversions of somehow randomly chosen CpGs can't be guaranteed
to reflect real life methylation patterns. Until the rules for positional
methylation are more fully understood, attempts to generate randomised
data are likely to be influenced by artefacts of the process used.

[rskr]

Originally Posted by aniruddha.otago

Hi rskr,

We never really thought of trying to do that. We were attempting
to see the performance with real RRBS data which we did. I'm not
sure that going to the trouble of trying to generate random CpG fragments
would have achieved very much at all. Any attempt to 'randomise' the
C/T conversions of somehow randomly chosen CpGs can't be guaranteed
to reflect real life methylation patterns. Until the rules for positional
methylation are more fully understood, attempts to generate randomised
data are likely to be influenced by artefacts of the process used.

It may true that the distribution of CpG methylation is potentially not random. However simply calculating unique matches doesn't tell you anything about the performance of the mapper, which is what you claimed to have measured. It may very well be that the correct mapping was indeed ambiguous, and you don't know. If you can't model the CpG methylation, then you can't claim to have measured the performance of the mapper.

[aniruddha.otago]

Originally Posted by rskr

It may true that the distribution of CpG methylation is potentially not random. However simply calculating unique matches doesn't tell you anything about the performance of the mapper, which is what you claimed to have measured. It may very well be that the correct mapping was indeed ambiguous, and you don't know. If you can't model the CpG methylation, then you can't claim to have measured the performance of the mapper.

it is done on a reduced represented genome. so, say there is a fragment of 100 bp and 8 reads uniquely mapped to that. mind you each fragment and read has to have a CGG or TGG (MspI site) at the start to be able to map. say, these 8 reads has total 16 CpG site. so, we counted this as a no of unique aligned CpG site. we did make commnet with a figure that even for the same region different programs showed different mapping. However, randomising CpG site didnt seem appropriate to us in the context of this paper. "It may very well be that the correct mapping was indeed ambiguous, and you don't know". If thats the case then no point doing any other step, because simply it is not giving you correct information. The aligners choosen here are widely used in the field and are well established to yield reliable results and we have no reason to believe that the correct mapping is ambigious.
we wanted to give a feel to the molecular biologists that if you have a RRBS dataset and use this widely used aligners, this is what you can get and what are the aspects one should be aware of. and also, a biologists is more likely to will work with real sample to analyse, not on "randomised CpG data".

[rskr]

Originally Posted by aniruddha.otago

... we wanted to give a feel to the molecular biologists that if you have a RRBS dataset and use this widely used aligners, this is what you can get and what are the aspects one should be aware of. and also, a biologists is more likely to will work with real sample to analyse, not on "randomised CpG data".

That's fine except I gather that your data is already out of date in technology terms(GAIIx), and wasn't very good to begin with, since you ended up trimming to make the mapping rates go up, indicating the quality dropped severely near the ends. Is that because of the RRBS protocol or something else? And as you mentioned you really don't have a model for methylation so there isn't any reason to believe that your results would be reproducible.