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===Filter by Mapping=== *All filtering was done in genemapster at </media/3TB_slot2/cjwei/Fate_Mapping/pp_design/ppDesigner_20140922> in each corresponding folder *For regular probes, we don't have to worry about strand specificity (i.e. there is no conversion in bases of sequence depending on the strand). Consequently, we can run the bash script: [[File:cw_20140922_1_filter-by-mapping.sh]] that performs the following steps 1) Creates simulated paired-end reads (one for each capture arm) from the original ppDesigner output using the perl script: [[File:cw_20140922_Probe_size.txt]] 2) We then verify that the capture arms map to the reference hg19 genome accordingly using Bowtie2 3) Once we have the output of Bowtie2 in sam format, we then want to filter out only those reads that mapped concordantly with both pairs mapping uniquely. Bowtie2 uses the tag "XS:i:" to denote unique mapping. Hence, we use samtools to filter only reads that mapped concordantly (using -Shf 0x2). Then we used grep to filter out any reads that had the "XS:i:" tag. We also remove header lines that start with "@." Finally, I used a python script from online that removes any orphan reads that mapped uniquely but the mate does not. This script is: [[File:cw_20140922_Extract_uniquely_mapped.txt]] 4) Next we extract only the read names from the sam file (i.e. extract only the targetIDs, one for each mate) using the script: [[File:cw_20140922_Sam2read-name.txt]] 5) We then extract only probe lines from the original ppDesigner output that were listed in the read names that passed the filter by mapping by using the custom script: [[File:cw_20140922_Merge_probe_files.txt]] *For bisulfite probes, we add a step in the beginning to divide the ppDesigner output into two files based on whether the probes target the Watson or the Crick strand. To accomplish this, I used the script: [[File:cw_20140922_Probe_split.txt]]. However, once we do that, we perform the subsequent steps listed above (steps 2-5) the same but for each strand separately then we combine all these in the end. The bash script that performs the steps is [[File:cw_20140922_1_filter-by-mapping-BSPP.sh]] *The resulting probe file after filter by mapping are as follows: <u>Bisulfite Probes</u> [[File:Hg19_2nt_ms_BSPP_20140922.txt.C.only_unique_alignment.txt]] [[File:Hg19_2nt_ms_BSPP_20140922.txt.W.only_unique_alignment.txt]] [[File:Hg19_3nt_ms_BSPP_20140922.txt.C.only_unique_alignment.txt]] [[File:Hg19_3nt_ms_BSPP_20140922.txt.W.only_unique_alignment.txt]] [[File:Hg19_4nt_ms_BSPP_20140922.txt.C.only_unique_alignment.txt]] [[File:Hg19_4nt_ms_BSPP_20140922.txt.W.only_unique_alignment.txt]] <u>Regular Probes</u> [[File:Hg19_2nt_ms_20140922.txt.only_unique_alignment.txt]] [[File:Hg19_3nt_ms_20140922.txt.only_unique_alignment.txt]] [[File:Hg19_4nt_ms_20140922.txt.only_unique_alignment.txt]]
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