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===Run ppDesigner=== *'''This part of the protocol has to be run on genome-miner, as Unafold is only installed there''' *I used ppDesigner v2 for this run (this can be found in genome-miner at </home/samchiang/Chris/software/ppDesigner_BSPP_v2.0/>) and can also be downloaded here [[File:cw_2014-09-22_PpDesigner_BSPP_v2.0.zip]] *In order to run ppDesigner for <u>bisulfite probes</u>, we perform the following steps (once I have the targetFiles for each type of microsatellite target): 1) Set the min/max melting temperature accepted for probes to 50/65 in the probe_parameters.pl file in ppDesigner by changing to the following lines: our $primerMaxTm = '''65'''; our $primerMinTm = '''50'''; 2) Use the paramters in the following example jobFile. These parameters are necessary as padlock probes targetting bisulfite converted DNA require longer probe arms for higher specificity. In addition, we have to also indicate that these probes are bisulfite probes [[File:cw_2014-09-22_jobFile_mod_90-110.txt]] 3) We then call and run ppDesigner using the following example command: /home/samchiang/Chris/software/ppDesigner_BSPP_v2.0-BSPP-params/src/ppDesigner.pl jobFile_mod_90-110.pl > hg19_2nt_ms_BSPP_20140922.txt *In order to run ppDesigner for <u>regular probes</u>, we perform the following steps (once I have the targetFiles fore ach type of microsatellite target): 1) Set the min/max melting temperature accepted for probes to 50/65 in the probe_parameters.pl file in ppDesigner by changing to the following lines: our $primerMaxTm = '''65'''; our $primerMinTm = '''55'''; 2) Use the paramters in the following example jobFile. These parameters are necessary as padlock probes targetting bisulfite converted DNA require longer probe arms for higher specificity. In addition, we have to also indicate that these probes are bisulfite probes [[File:cw_2014-09-22_regular_probes_JobFile_mod_90-110.txt]] 3) We then call and run ppDesigner using the following example command: /home/samchiang/Chris/software/ppDesigner_BSPP_v2.0/src/ppDesigner.pl jobFile_mod_90-110.pl > hg19_4nt_ms_20140922.txt *I ran each of these commands separately for each type of microsatellite (2nt, 3nt, 4nt) and the raw output files from ppDesigner as as follows: <u>Bisulfite Probes</u> [[File:Hg19_2nt_ms_BSPP_20140922.txt]] [[File:Hg19_3nt_ms_BSPP_20140922.txt]] [[File:Hg19_4nt_ms_BSPP_20140922.txt]] <u>Regular Probes</u> [[File:Hg19_2nt_ms_20140922.txt]] [[File:Hg19_3nt_ms_20140922.txt]] [[File:Hg19_4nt_ms_20140922.txt]]
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