Chris:LabNotes/FateMapping/Calendar/2015/2015-7-2

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Revision as of 20:18, 2 July 2015 by >Cjwei (Created page with "=Verify filtered 20140922 probes= ==Background== *Previously, we decided on filtering the probes designed on 20140922. This was done by taking the top 2000 probes from either...")
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Verify filtered 20140922 probes

Background

  • Previously, we decided on filtering the probes designed on 20140922. This was done by taking the top 2000 probes from either gDNA, gDNA MDA, or C1 single cell capture then removing the very top 20. This is described on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2015/2015-5-23>. Theoretically, by cherry picking the top probes and removing the very top, we should get much better probe capture/more even bias compared to the larger probe set.
  • However, upon probe capture on new BA17 C1 single cell amplicons done on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2015/2015-6-15>, we did some analysis on the sequencing data and it looks like bias actually turned out to be worse.
  • We will be verifying probe design today. One potential concern is that the probes may have been designed/ordered differently than originally done, so we will be looking at the scripts that Dr. Zhang used to design these probes and also the final ordering file sent to CustomArray
  • As a reminder, the two different probe sets were used on different single cell amplicons from BA17 (2015_02_26 C1 run):
Well #     Probe Set     Capture Link
G1         Original      http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2015/2015-3-13 (Strip A = Hemo, Strip B = Phusion)
H1         ""            ""
A2         ""            ""
B2         ""            ""
C2         ""            ""
D2         Filtered      http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2015/2015-6-15
E2         ""            ""
F2         ""            ""
G2         ""            ""
A3         ""            ""

Verify Scripts

  • Below are the scripts used when Dr. Zhang finalized the filtered probe sets for order at Custom Array:
File:Cw 20150702 Probe2padlock13May2014V4.txt     20150523 New Probes (new set of 8000 not used in this analysis, v4)
File:Cw 20150702 Probe2padlock13May2014V6.txt     Filtered Hemo Probes (v6)
File:Cw 20150702 Probe2padlock13May2014V8.txt     Filtered Phusion Probes (v8)
  • Below is the script used to make the original probe order from 20140922:
File:Probe2padlock30Sept2014V6.txt
  • There are a few differences between the new probes from teh old probes based on the scripts (v6 and v8 vs. the original probe script)
    • LinkerL is 4nt longer (with TATT added) for the new probes
    • LinkerR is 8nt longer (with CAGGCAGA added)
    • The UMI sequence is 6nt vs the original 8nt used in the original probe
    • For the new probes, the maxH1H2Len parameter is 4nt longer than the original (maxH1H2Len=46). Because the original ppDesigner parameter of H1_plut_H2_Len was original set to 42nt for the original 20140922 probes along with the new filtered probes (which was just a copy and paste of lines from the original probe file), there is a 4nt poly-T filler within every new padlock probe designed

Comparing UMI Capture