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==== Step 2.3: Running bismark methylation_extractor ==== * In directory: NP.RRBS.Trim1_directional/Bismark-run.directional * I used default set up '''Run.bismark.methylationExtractor.sh''' for f in ./*bismark.sam do /home/nplongth/softwares/bismark_v0.12.2/bismark_methylation_extractor -s --comprehensive $f done * I simply use awk command to get chromosome position and start position in column $3 and $4, respectively in CpG_context.XXXX._bismark.txt files. Then sort out the unique line to get minimum 1x unique CpGs Ex. awk '{print $3"\t"$4}' CpG_context_apr10_1to50_no1.txt_trimmed.fq_bismark.txt | awk '{if($1~/chr/) print $0}' | sort -u | wc -l * I also need to run with bowtie2 to compare the result with bowtie1 * Need to double check and learn bismark in great details. * There is another point I need to add to libraries in pool1 (green highlighted). When I did PAGE size-selection, the gel was badly stuck to the cassette, and I may not cut at the right size and I lost some lane. The yield of this pool was very low. I am not sure if this is the reason that I got unique CpG lower than other pools. ===== 2014-05-27 : Response from Dr. Zhang ===== :- I used bowtie for mapping and haven't tried bowtie2. I can try it out later to see the different. :''Itโs okay for now. In the future, I think we want to run adaptor trimming, then map the reads using our own bisReadMapper'' <br> :- I am not very sure if I should treat RRBS data as directional or non-directional. I have tried both ways. It turned out that non-directional option has lower mapping rate. :''I believe itโs directional, as the adaptors were added prior to bisulfite conversion.'' <br> :- For bismark mapping, I allowed 1 non-bisulfite mismatch per read. I am not sure if this is too stringent. :''Should be fine for short reads. When have longer HiSeq reads in the future, you want to allow for 1 mismatch per 30bp.'' <br> :- I have not continued to extract methylation data into bed file and find out the coverage at different read depth yet. I need your suggestion before go to the next step. :''Please go ahead to extract the methylation information, and share the bed files with me. Iโd like to take a look by myself in next day or two.''
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