Chris:LabNotes/FateMapping/Calendar/2014/2014-9-22: Difference between revisions

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  Merge to determine intersection of BSPP and regular probes (targetting strands w/o C)*
  Merge to determine intersection of BSPP and regular probes (targetting strands w/o C)*
==Procedure==
==Procedure==
===Change targetFile to contain strands w/o C*===
===Change targetFile to contain strands w/o C===
*This step is performed for probe categories 1 and 2

Revision as of 00:27, 23 September 2014

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