Kun:LabNotes/ASE
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2010 <calendar> name=Kun:LabNotes/ASE format=%name/%year-%month-%day date=2010/02/01 view=oneyear </calendar> 2009 <calendar> name=Kun:LabNotes/ASE format=%name/%year-%month-%day date=2009/02/01 view=oneyear </calendar> 2008 <calendar> name=Kun:LabNotes/ASE format=%name/%year-%month-%day date=2008/02/01 view=oneyear </calendar> 2007 <calendar> name=Kun:LabNotes/ASE format=%name/%year-%month-%day date=2007/11/01 view=threemonths </calendar>
CES18bp probe design (Jan 09)[edit]
- Considerations of this probe set:
- Gap size increased to 18bp. The variant site is 13bp away from the ligation terminal, and 4bp away from the extension terminal.
- All probes designed for the sense strand.
- codes;
- 39766 probes were designed, only 8583 probes were selected to fit the space of the Jan09 order. I selected the subset of probes based on the following criteria:
- All probes on chromosome X and predicted/confirmed imprinted genes, 1061;
- For SNPs in other regions, folding energy of the target sequences (deltaG: -10 to -0.22), minor allele frequencies (0.19 to 0.5)
- Probe annotation file.
- Probe prep schema.
Design of the CES27K9bp probe set (Aug. 2008)[edit]
- This is the second probe set that has a gap larger than 1bp. The major differences between CES27K9bp and CES27K are:
- The gap size is 9bp, and the SNPs are 2bp away from the ligation arms (H1) and 6bp away from the extension arms (H2). The reason to place the SNPs further way from the extension arms is to increase the annealing stability of the extension arm with the extension of 6bp before the SNPs are filled. Hopefully this will reduce the allelic difference of circularization efficiency for the probes.
- The annealing temperature was increase to ~65C for the ligation arms and ~53C for the extension arms.
- Procedure for probe design:
- Map common SNPs (MAF>=0.05) to RefSeq genes: codes;
- Extract 50bp upstream/downstream sequences for the SNPs from the transcriptome: codes;
- Design capturing arms for each SNPs, check for the uniqueness of each captured target: codes;
- Convert the sequences for capturing arms into Agilent oligo libraries: codes
- Find the MAF and the distance to splicing junctions for each SNP (should have been included earlier): codes;
- Select a subset of probes in Excel based on:
- Tm(H1): 60-72C; Tm(H2): 50-62C;
- Distance to splicing junctions: at least 25bp;
- MAF: at least 0.075
- MW: probe=33KD; Solexa library=105KD (171bp)
- Probe annotation file.
- raw probe sequences.
- Capturing efficiencies based on end-sequencing. A more complete data set.
Design of the CES27K probe set[edit]
- This is the probe set following the CES22k set. The major differences are:
- The SNPs were chosen based on RefSeq gene instead of the knowngenes table in the GoldenPath database.
- SNPs with MAF>0.05 are included.
- I decided to extend the gap size from 1bp to 10bp, which will allow the exclusion of some non-specific circularization products.
- The capturing arms are Tm normalized with the exception of some SNPs in regions with extreme GC content.
- Codes for getting SNP flanking sequences
- Codes for generating capturing sequences
- Amplification adaptors: Version 6.
- Linker: Version 6, same as CES22k.
- Codes for assembling padlock probes
- Probe sequences
- Probe information