bicycle: bisulfite-based methylcytosine caller

[lipido]

Hi,
I wish to share with you a new BS-SEQ project: bicycle

bicycle (bisulfite-based methylcytosine caller) is a next-generation sequencing bioinformatics pipeline able to perform a full DNA methylation level analysis, as well as methylcytosine calling, for all of the cytosines that are present in one of the three known methylation contexts (CG, CHG, CHH), based on the directional Lister et al. MethylC-seq protocol and analysis procedure.

Please, take a look at the project homepage: http://sing.ei.uvigo.es/bicycle

[canadian_corvid]

Hi Lipido, I have a question about your tool, which looks to have some very nice features.

Is there support for bowtie2? Bowtie2 has the very useful --local alignment option, which I find valuable since I'm working on an amplicon resequening project. Alternately, does your "analyze-methylation" command accept a SAM file generated with bowtie2 (or any other alignment program for that matter) or can the alignment only be performed using the 'align' command from within bicycle?

[lipido]

Hi,
By now bicycle does not support changing the aligner out-of-the-box.

Altough we allow the user to change two parameters of the underlying bowtie, we force the remaining parameters to:
- Report only one alignment per read (With -M 1, if there are more alignments, use the XM flag in SAM). Reads with more than one alignment position will be filtered out in the next step. We have done this because in the Lister protocol there are several strict conditions (because the genomes are more simpler, since there are no Cytosines in the Watson ref. and there are no Guanines in the Crick ref.), so a given read is more easy to be aligned in several positions. So only non-ambiguous alignments should be considered.

I think that we can see if the --local alignment parameter of bowtie2 is compatible with -M 1 (report only one alignment). If so, I think that it could be easy to include it in further versions of bicycle.

The second solution is "hacking" the sam files. You can try to do that looking at the output directory of the project after align and see how bicycle names the sam files. You can try to replace them (with the same name) before running the analysis step.

In addition, I want to say that the analysis step was implemented with GATK framework. In fact, It is a GATK locus walker. We could provide this walker as a separated piece, but we have not documented this.

In summary, I think that we should explore the first way, so: Does bowtie2 allows -M 1 along with --local?

[xfliwz]

Hello lipido,
I want to use bicycle to analyze paired-end sequencing reads. As mentioned on the manual, the latest version supports paired-end reads. But I do not know how to set the options. So, could you show me some example for paired-end reads analysis?
Thanks for your attention!
Yours truly
xfliwz

[lipido]

Hello,

First, you have to put the .fastq files of the samples in subdirectories under your reads directory. For example (for one sample):

<reads_directory>
   sample-1/  <---- subdirectory
        fileA_1.fastq
        fileB_2.fastq

Then, you have to provide a SUFFIX for mate1 files (in this example "_1.fastq" should be sufficient) in the create-project command, with the -m parameter.

And finally, in the align command, remember to add the -I and -X params to specify the expected gap size. For example -I 0 -X 250.

Hope it works! Let me know if not.

[xfliwz]

Hello,
I used that in -m "_1.fastq" and it can create-project, but it throw wrong message when process reads-bisulfitation. The message are:

Sep 26, 2012 6:53:19 PM es.cnio.bioinfo.bicycle.operations.SampleBisulfitation computeSampleBisulfitation
INFO: Performing CtoT in-silico bisulfitation for /home/Project/bicycle-test/sample-1/chr10SimB_2.fastq......
Error during execution: String index out of range: 5
java.lang.StringIndexOutOfBoundsException: String index out of range: 5
at java.lang.String.charAt(String.java:686)
at es.cnio.bioinfo.bicycle.operations.SampleBisulfitation.computeSampleBisulfitation(SampleBisulfitation.java:78)
at es.cnio.bioinfo.bicycle.cli.ReadsBisulfitationCommand.executeImpl(ReadsBisulfitationCommand.java:49)
at es.cnio.bioinfo.bicycle.cli.ProjectCommand.execute(ProjectCommand.java:52)
at es.cnio.bioinfo.bicycle.cli.CLIApplication.run(CLIApplication.java:79)
at es.cnio.bioinfo.bicycle.cli.Main.main(Main.java:29)

[lipido]

Mmmmm..., It seems that you have used the -b option in the reads-bisulfitation command. Does your reads where generated using barcodes? If you do not know this, do not use -b.