Chris:LabNotes/FateMapping/Calendar/2014/2014-9-22

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Revision as of 22:59, 23 September 2014 by >Cjwei
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Probe Design (filtered bisulfite and regular probes)

Background

  • Previously we found that the regular probes designed on 5/12 contained some capture arms that had very low alignability scores (i.e. will map to multiple places in the reference genome). This would consequently cause non-specific binding and may in turn be the cause of the very broad smear we're seeing in the probe capture gels. Because of this, I'll be redesigning the probes and applying a post-ppDesigner quality filtering.
  • In addition, after talking with Dr. Zhang, we want to also begin to look at DNA methylation at the same time as microsatellite data in order to obtain both phylogenetic and epigenetic data from each single cell. In order to do this, we'll need to perform bisulfite conversion on single cell DNA and perform probe capture. Consequently, I'll also be making another set of BSPP probes for bisulfite capture of microsatellite sites.
    • Because of the bisulfite conversion, all unmethylated C's will turn to U's, which in turn will be represented as T's after amplification. Consequently, when designing probes, we want to avoid the strand containing cytosines
  • The final padlock probes designed will fall into one of the following four categories (depending on the availability of space on the chip, we can order some of the following):
1) Bisulfite Padlock Probes (targetting strands that do not contain cytosine)
2) Regular probes (targetting strands that do not contain cytosine)
3) Regular probes (targetting either strand without preference for non-cytosine strand)
4) Intersection of BSPP and regular probes targetting strands that do not contain cytosine (for comparison purposes)
  • The general procedure is as follows (* indicates the steps may be skipped depending on the type of probes being produced)
Change targetFile to contain strands w/o C*
     |
     V
Run ppDesigner
     |
     V
Map against reference (BSPP strand specific mapping, regular map against genera hg19 genoem)
     |
     V
Merge to determine intersection of BSPP and regular probes (targetting strands w/o C)*

Procedure

Change targetFile to contain strands w/o C

  • This step is performed for probe categories 1 and 2
  • Each microsatellite target derived from the UCSC genome browser contains the sequence microsatellite subunits along with the number of times that subunit is repeated (ex: 10xAG). However, because we want to run bisulfite conversion prior to probe capture, we want to take into account that C's that are unmethylated will be converted to U's that would consequently be converted to T's after amplification. Consequently, we want to avoid the strand containing C's in the MS target and use the opposite strand.
  • To do this, we use the fact that the MS targets listed in the bed file refer to the Watson (+) strand of DNA in the reference. So, if there is a C in the MS target sequence (ex: 10xAC), we want to choose the Crick strand (-). If there is a G in the MS target sequence (ex: 10xAG), we want to choose the Watson strand (+). If there are neither C or G in the MS target, we can allow ppDesigner to choose the strand, and if there are both C and G in the MS target, we have to throw out the target altogether.
  • To accomplish this, I wrote a script <File:Cw 2014-09-22 Microsat filter all.txt> that takes in the bed file containing all MS targets <File:Cw 2014 09 22 Hg19 2nt-3nt-4nt MS location.txt> and outputs three targetFiles (one for each type of MS, i.e. 2nt, 3nt, 4nt) with the fourth column of the targetFile. The command to run this is as follows:
perl microsat_filter_all.pl hg19_2nt-3nt-4nt_MS_location.txt
  • The following are the resulting MS targetFiles that will be inputted into ppDesigner:
File:Cw 2014-09-22 targetFile 2nt.txt
File:Cw 2014-09-22 TargetFile 3nt.txt
File:Cw 2014-09-22 TargetFile 4nt.txt

Run ppDesigner

  • 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 bisulfite probes, we perform the following steps (once I have the targetFiles for each type of microsatellite target):
    • 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;
    • 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
    • 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