Chris:LabNotes/FateMapping/Calendar/2015/2015-5-23
Jump to navigation
Jump to search
Probe Design[edit]
Background[edit]
- We want to make three different probe sets from this probe 20150523 design:
1) Cherry picked Hemo probes from 20140922 probes 2) Cherry picked Phusion probes from 20140922 probes 3) New 20150523 probe design with adjusted melting temperatures
Procedure[edit]
Cherry picked Hemo probes from 20140922 probe set[edit]
- We wanted to pick out the best probes based on #UMI/target from the Hemo reactions. In order to do this, we took the average of the #UMI/target for all previous reactions in strip C in the March 13 probe capture <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2015/2015-3-13>, downsampled to the same number of reads mapped. The raw counts is found in the file in genemapster </media/3TB_slot2/cjwei/Fate_Mapping/pp_sequencing/150428_MiSeq/2_determine_seq_effort/summary_UMI/downsampled_mapped/Hemo.counts>.
- The excel file with the selection/ranking of probe captures is contained in File:Cw 20150523 Hemo average.xlsx
- We then chose the top 2000 probes but deleted the very top 20 probes in order to decrease probe capture bias. The final range for #UMI/target is ~100 to 5. This was done in genemapster </media/3TB_slot2/cjwei/Fate_Mapping/pp_design/ppDesigner_20140922/regular_probes_noC/filtered_Probes_20150523>
Cherry picked Phusion probes from 20140922 probe set[edit]
- We did the same calculation as above with the average of #UMI/target for all previous reactions in strip D in the March 13 probe capture, downsampled to the same number of reads mapped. The raw counts is found in the file in genemapster </media/3TB_slot2/cjwei/Fate_Mapping/pp_sequencing/150428_MiSeq/2_determine_seq_effort/summary_UMI/downsampled_mapped/Phusion.counts>.
- The excel file with the selection/ranking of probe caputures is contained in File:Cw 20150523 Phusion average.xlsx
- We then chose the top 2000 probes but deleted the very top 20 probes in order to decrease probe capture bias. This was done in genemapster </media/3TB_slot2/cjwei/Fate_Mapping/pp_design/ppDesigner_20140922/regular_probes_noC/filtered_Probes_20150523>
New 20150523 probe design[edit]
- We now wanted to design a new set of probes for an expected set of targets. These probes will be designed using a modified version of ppDesigner that allows for finer tuning of the capture arm melting temperatures. Previously, the melting temperatures were set with global parameters $primerMaxTm/$primerMinTm in the probe_parameters.pl file. It would result in probes that had ligation/extension arms that were generally between the melting temperatures of 55/65C.
- However, Dr. Zhang noticed that the best probe captures happened when the ligation arm melting temperatures were higher (along with extension arm Tm being ~5C lower than ligation arm Tm). Consequently, we want to edit ppDesigner in order to set the melting temperature of ligation arms to anywhere between 66 and 72. Then, we want to control the melting tmeperature of the extension arms to be within 4-8C of the ligation arm Tm. This was all done by editing the get_probes.pl script within ppDesigner. The final ppDesigner zipped program is found in File:Cw 20150523 ppDesigner BSPP v2.0 edit.zip
- Note:Because we had to increase the melting temperature of the probes, I also increased the total length of the two capture arms H1_plus_H2_len to 48 from 42bp in the jobFile.
- The final ppDesigner run was in genome-miner </home/cjwei/Fate_Mapping/pp_design/ppDesigner_20150523/>.
- We then wanted to filter by alignment and alignability using the same procedure as <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2014/2014-9-22> and then wanted to only choose the probes that were not also covered by the cherry-picked 20140922 probes. This was all done in genemapster at </media/3TB_slot2/cjwei/Fate_Mapping/pp_design/ppDesigner_20150523>
Results[edit]
- Total probe order: 12472 probes available for 12k Custom Array Chip
- The file for the top 1980 Hemo probes from the 20140922 ppDesigner run is found in: File:Hg19 regular probes noC 20140922.hemo.txt
- The file for the top 1980 Phusion probes from the 20140922 ppDesigner run is found in: File:Hg19 regular probes noC 20140922.phusion.txt
- The file for the top 8512 2nt-6nt probes (not in the previous two cherry-picked files) can be found in: File:Hg19 probes 20150523.txt
- The probes ordered have a UMI of length 6, NOT length 8 as was the previous 9/22 probe set