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==Probe design== ===Human DMR 460k set (Jan 2014)=== * Set name: '''HOTSPOTS''' (Hypersensitive and Open Tissue SPecific Optimized Target Sites) * Target design and description of HMM segmentation: http://genome-tech.ucsd.edu/LabNotes/index.php/Dinh/Dinh_2013/NOTES/2013-12-13 * Probes design with new neural network: http://genome-tech.ucsd.edu/LabNotes/index.php/Dinh/Dinh_2014/NOTES/2014-1-9 ** 375,011 probes cover high methylation variable regions ** 22,895 probes added to cover genes and lincRNAs ** 15,000 high methylation variable probes from TFBS/DHS/SE overlapping regions. * Summary of final probes designed: http://genome-tech.ucsd.edu/LabNotes/index.php/Dinh/Dinh_2014/NOTES/2014-1-9#Summary_of_probes ===Human DMR 330k set (July 2011)=== *This is also known as the Human DMR 220k expansion set. *[http://genome-tech.ucsd.edu/LabNotes/RobertsLabNotesSupplement/DMR330k_probeInfo_efficiencies.txt.gz Probe annotation (including efficiencies based on PGP1iPS data)] ===Human DMR 220k expansion set (Dec 2010)=== *[[User:Expresso/Cpg220kProbeSetExpansion|This probe set]] was designed by Robert to complement the DMR220k set. It covers low-efficiency regions in s5-7, plus all RefSeq TSS: [http://genome-tech.ucsd.edu/LabNotes/RobertsLabNotesSupplement/NonOverlappingGeneratedProbes.tar.gz Non-Overlapping Generated Probes] LibraryName OLID DMR220kExp1 0320561 DMR220kExp2 0320571 DMR220kExp3 0320581 ===Human DMR 220k probe set (May 2010)=== *This probe set was designed based on the following targets: **core: ***~20,000 DMRs provided by Andy Feinberg's group at JHU. These DMRs were identified by the CHARM analysis; ***~30,000 targets provided John Stamatoyannopoulous's group at UW. These targets were mainly selected based on DNase I sensitive regions. **imprinted: Imprinted genes (Robert). **h1imr90enh: Enhancers in H1 and IMR90 cell lines (Robert). **salkaml: Additional DMRs based on Salk's BisSeq data and AML data (Robert). *Parameter file for probe design. *[[Media:probe2padlockCpgLibMay10.txt| Probe assembly script.]] *Probes designed and ordered for each subset: Designed Ordered Core.50k 170567 170567 H1IMr90Enh 8023 8023 Imprinted 3304 3304 SalkAml 52080 38106 Total: 233974 220000 *All 220k probes were split into four 55k libraries: **[[Media: DMR220k_probeSeq_a.txt.gz | DMR220k_a]]: OLID 0284601 **[[Media: DMR220k_probeSeq_b.txt.gz | DMR220k_b]]: OLID 0284591 **[[Media: DMR220k_probeSeq_c.txt.gz | DMR220k_c]]: OLID 0284581 **[[Media: DMR220k_probeSeq_d.txt.gz | DMR220k_d]]: OLID 0284571 *The probes were re-grouped based on the relative capturing efficiencies: **[http://genome-tech.ucsd.edu/LabNotes/upload/large_files/DMR220k_norm_probes.xlsx Probe efficiency and grouping info file.] **DMR220k.norm.s1_6500 0294691 **DMR220k.norm.s2_6500 0294701 **DMR220k.norm.s3_55k 0294711 **DMR220k.norm.s4_55k 0294721 **DMR220k.norm.s5_55k 0294731 **DMR220k.norm.s6_27k 0294741 **DMR220k.norm.s7_27k 0294751 ===Mouse probes (Apr. 2009)=== *This is a probe set designed for JHU/VCU. ([[Media:probe2padlockCpgLibFeb09.txt | probe2padlockCpgLibFeb09.pl]]) Set ID # Targets #probes JHU_mm9 88 8,682 ChrX_mm9 893 35,291 ChrY_mm9 15 521 Imprint_mm9 114 6,786 miRNA_mm9 493 27,360 GO_mm9 1170 *First probe set ([[Media:CpgMm9A-H1H2.txt.gz |CpgMm9A]]) ordered: *55,000 probes. **JHU_mm9 **ChrY_mm9 **Imprint_mm9 **ChrX_mm9 **miRNA (first 3720 probes) ===Agilent probes, V3 (Feb. 2009)=== *Targets **16,379 T-DMRs identified by Irizarry et al. Nature Genetics 41:178-86, 2009 **SwitchGear TSS that overlap with CCDS first exons (-400bp to 200bp relative to TSS). Only 775 targets on chrX were included. **179 known or predicted imprinted genes *Degenerate probes: [http://genome-tech.ucsd.edu/LabNotes/upload/large_files/CpgProbes_Jan-chrX_Feb_09_combined-H1H2.xlsx Probe Information] [[Media:probe2padlockCpgLibFeb09.txt | probe2padlockCpgLibFeb09.pl]] [[Media:Amplifiable_MIP_design_KZ_v11.doc|Probe prep schema]]. **69,263 were designed. **Divided into four subsets based on dG: **Set I: dG <= -20; 461 (19 are too long) => 1147 non-degenerate probes **Set II: -20 < dG <= -8; 15,871 (1212 are too long) => 24,322 non-degenerate probes **Set III: -8 < dG < 0; 53,033 (1892 are too long) => 70,294 non-degenerate probes (IIIa: top 15,294 probes; IIIb: bottom 55,000 probes) **Set IV: dG > 0; 1,122 (32 are too long)=> 1,313 non-degenerate probes **The total number of non-degenerated probes is 97,076, which capture 67,332 targets. **The top 55,000 oligos (-8<dG<-2) are in CpgProbes_Jan_55kA; **The remaining 42,076 oligos are in CpgProbes_Jan_55kB. ***9136 Exome-March-09 non-unique probes (amplified with the V4 primers) were also included in this library. ***The remaining spaces were filled by all probes in Set I and some in Set II. **103nt, MW=34kD ===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?[[Media:getCpgProbes.txt|Code.]] #The codes for the search of capturing sequences were completely rewritten. ##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. ##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). ##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. ##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. ##The candidate sets are then sorted based on the score, and the one with the best score was selected. #To evaluate the specificity of capturing sequences, the counts of all 12-mers in both strands of the [[Media:bisCvt.txt|bisulfite converted]] genome were [[Media:oligoSamp.txt|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 15,998 973,377/924,553 799,818/767,129 82.1%/83.0% Chromosome 20 11,651 654,755/693,920 576,059/550,144 83.0%/84.0% Additional space for other targets: #Key genes in maintaining pluripotency or differentiation. 2000bp up/down-stream of the Transcription Start Sites (TSS, based on SwitchGear TSS), ES-Fib-diff: 780 probes #Genes suggested by the Hochedlinger lab (MEG3,SNRPN,CDX1,HAND1,PAX6,FGF7) KH: 293 probes. [[Kun:LabNotes/CpgSeq/Cpg30k_selected_genes_list|Gene list of #1 & #2.]] #ENCODE regions, Stanf promoters: ENm004: Chr22 151 probes chr22:30,133,954-31,833,953 ENm005: Chr21 271 probes chr21:32,668,237-34,364,221 ENm006: ChrX 307 probes chrX:152,767,492-154,063,081 ENm007: Chr19 276 probes chr19:59,023,585-60,024,460 ENm010: HOXA cluster 135 probes chr7:26,924,046-27,424,045 ENm011: 1GF2/H19 188 probes chr11:1,699,992-2,306,039 ENr123: Chr12 10 probes chr12:38,626,477-39,126,476 ENr333: Chr20 157 probes chr20:33,304,929-33,804,928 Added: ES-Fib-diff 776 (duplicate probes) Total 30,000 Split into three sets: merge chr12 and chr20 probes, sorted by H1/H2 specificity [[Media:cpgMIP30kV2.1b.txt|cpgMIP30kV2.1: low score]] [[Media:cpgMIP30kV2.2b.txt|cpgMIP30kV2.2: medium score]] [[Media:cpgMIP30kV2.3b.txt|cpgMIP30kV2.3: high score + other subsets]] [[Media:cpgMIP30k_probeInfo.txt|Probe information]] [[Media:cpgMIP30k_2008-06-12-read-counts.txt|Capturing Efficiencies based on end-sequencing.]] ====Additional probes for normalizing the cpgMIP30k set==== *A cpgMIP30k captured library was end-sequenced on 06/12/08, 10364/10582 targets showed up at least once. So I sorted all 10582 probe templates in the order from low abundance to high (abundance for the ones did not showed up were considered 0). These templates were converted to probes with probe2padlockCpgLibV2.pl, and split into four sets (the last one not synthesized). #cpgMIP30kNormL: 4677 probes, abundance: 0-10; the remaining space was filled with 5323 CES22k set on the low end of the distribution on gDNA capture (based on Hues62/63 data on 06/12/08). #cpgMIP30kNormM: 9,999 probes (one included twice), abundance: 11-80 #cpgMIP30kNormH: 10,000 probes, abundance: 80-881 ===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 [[Media: getCpGPrimers.txt |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. [[Media:primer2padlockCpgLibV1.txt|codes]]. <u>Some statistics</u> 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 **[[Media:Cpg28kV1_probes.gz|Probe information file.]] **[[Media:Cpg28kV1.txt|Probe raw sequences.]] **[[Media:cpgMIP28k_2008-05-14-read-counts.txt|Capturing Efficiencies based on end-sequencing.]] **ssDNA probes: len=102nt, MW=31KDa. ===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: [[Media: CpG_primer2padlock_v2.txt | first 24 primers]]; [[Media: CpG_primer2padlock2_v2.txt | second 12 primers]]. Here is the new [[Media: CpG_probes_v2.txt | probe file]].
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