Kun:LabNotes/CpgSeq: Difference between revisions

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====cpgMIP30k probe set====
====cpgMIP30k probe set====
Two chromosomes enriched in genes differentially expressed between hES/iPS and fibroblasts.
Two chromosomes enriched in genes differentially expressed between hES/iPS and fibroblasts.
   Chromosome 12       16,998 probes
                  # probes  total bps covered  CGI covered    %CGI covered
   Chromosome 20       12,250 probes
   Chromosome 12   16,998       783,395          771,362        90.6%
   Chromosome 20   12,250       531,943          523,861        85.3%
Additional space for other targets:
Additional space for other targets:
#Key genes in maintaining pluripotency or differentiation. 500bp up/down-stream of the Transcription Start Sites (TSS, based on SwitchGear TSS), ES-Fib-diff: 287 probes
#Key genes in maintaining pluripotency or differentiation. 500bp up/down-stream of the Transcription Start Sites (TSS, based on SwitchGear TSS), ES-Fib-diff: 287 probes
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   ENm011:      175 probes
   ENm011:      175 probes
   ENr333:        89 probes
   ENr333:        89 probes
   Still a space for 94 probes.
   Still a space for 94 probes, added the first 94 probes for ENm006 (chr X).


===Agilent probes, V1===
===Agilent probes, V1===

Revision as of 05:01, 5 May 2008

2008 <calendar> name=Kun:LabNotes/CpgSeq format=%name/%year-%month-%day date=2008/02/01 view=oneyear </calendar> 2007 <calendar> name=Kun:LabNotes/CpgSeq format=%name/%year-%month-%day date=2007/11/01 view=threemonths </calendar>

Probe design

Agilent probes, V2 (April 2008)

  • Why redesigning probes?
    • V1 design was based on Primer3. Many constrains in Primer3 are probably either unnecessary or inappropriate for padlock probes.
    • We would like to search for an optimal (maximal coverage, minimal number of CpG in H1 & H2) set of probes covering a CpG island. It is hard to integrate Primer3 into the search of such an optimal set.
    • The capturing sequences should be as specific as possible. They don't have to be completely unique however, since a low percentage of non-specific capture is acceptable.
  • What are the differences in the V2 algorithm?Code.
  1. The codes for the search of capturing sequences were completely rewritten.
    1. The algorithm first search for all oligos with appropriate length and Tm, then look for pairs with appropriate gap size among all pair-wise combinations of these oligos.
    2. These pairs were assigned scores based on criteria including Tm, size, gap size, number of CpGs and a frequency score based on word statistics (word size K=12).
    3. Because the number of such pairs could be very large, the target region was then divided into a number of 10-bp bins, and only the pair with the best score in each bin was selected for the next round of optimization.
    4. These pairs are then assembled into sets. These sets cover every combination of the pairs. Each set has a score, which is based on the score of each pairs, plus the coverage of all pairs in the set.
    5. The candidate sets are then sorted based on the score, and the one with the best score was selected.
  2. To evaluate the specificity of capturing sequences, the counts of all 12-mers in both strands of the bisulfite converted genome were calculated, and saved as a data file. This file was loaded into the memory during probe design. The frequency score of each sequence is a weighted sum of all 12-mer sub-sequences. This calculation has a directionality, because the specificity for the sub-sequence close to the gap (for ligation or extension) should be higher. So the weight for the first 12-mer next to the gap is 1, and it is reduced by 25% per base as the sub-sequence moves outwards. I also added a hash table as a buffer so that the same oligo doesn't have to be calculated over and over again.

cpgMIP30k probe set

Two chromosomes enriched in genes differentially expressed between hES/iPS and fibroblasts.

                  # probes  total bps covered   CGI covered    %CGI covered
  Chromosome 12    16,998        783,395          771,362         90.6%
  Chromosome 20    12,250        531,943          523,861         85.3%

Additional space for other targets:

  1. Key genes in maintaining pluripotency or differentiation. 500bp up/down-stream of the Transcription Start Sites (TSS, based on SwitchGear TSS), ES-Fib-diff: 287 probes
  2. ENCODE regions, Stanf promoters:
    1. ENm010: HOXA cluster 135 probes
    2. ENm011: 1GF2/H19 188 probes
    3. ENr333: chromosome 20 157 probes

After removing redundant probes.

  ES-Fib-diff:  277 probes
  ENm010:       117 probes
  ENm011:       175 probes
  ENr333:        89 probes
  Still a space for 94 probes, added the first 94 probes for ENm006 (chr X).

Agilent probes, V1

  • Several considerations in this design include:
    • Degenerate bases: I allowed at most two degenerate bases per primer. To convert PCR primers to padlock probes, multiple probes will be designed for one primer pair in order to cover all combinations.
    • Gap size: The range of amplicon size is 225-275bp, so the gap size is approximately 185-225bp.
    • Amplification adaptors: Version 4; Linker sequences: Version 7
   AP1: GTAGACTGGAAGAGCACTGTT  V4
   AP2: GATCGGATACGCATGAGGCTA  V4
   Linker: GTTGGAGGCTCATCGTTCCTATTCAGCTGCAGATGTTATCGAGGTCCGAC
           ------                  ----                ------
           Mme I                   Alu I                Mme I
   
  • Here is the perl code for primer design. The input file is cpgIslandExt.txt downloaded from UCSC GoldenPath database.
  • Conversion of PCR primers to padlock probes: AP1 + revcomp(FP) + linker + RP + AP2. codes.
  Some statistics
  Total probes:                     210,498
  Total CpG islands:                 23,496
  Total non-overlapping regions: 12,037,010 bps
  Total captured sequences:      13,291,608 bps
  ===CpgMIP28k set===
          probes                   CpG islands
  Chr6:   9,875          3,028    (1103/1251, 559kb/940kb)
  Chr20:  6,197          1,930    (695/779, 358kb/613kb)
  Chr21:  2,619            809
  Chr22:  5,358          1,664    (622/716, 310kb/562kb)
  Chr19:  first 3,857

Column synthesized probes (36)

  • Kun:LabNotes/CpgSeq/2007-10-12: probe = revcomp(RP) + linker + FP
  • Just realized that there was a mistake in probe design (11-20-2007). I assumed that after bisulfite treatment, the two strands are still complementary, which is not true. The probes have to be designed based on one strand only. If the forward PCR primers contain no "C" and the reverse primers contain no "G", that means the target strand is the forward strand. In that case, the padlock probes should be: revcomp(FP) + linker + RP. Here are the revised perl scripts: first 24 primers; second 12 primers. Here is the new probe file.

Library construction