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===Design of new probe sets=== *Blood LMS BSPP probes. **Targets: [[Media:whole_blood_LMS_clusters_input_plus.txt]]; [[Media:whole_blood_LMS_clusters_input_minus.txt]] **Job files: [[Media:jobFile_Blood_LMS_100_plus.txt|jobFile_Blood_LMS_100_plus.pl]];[[Media:jobFile_Blood_LMS_100_mius.txt|jobFile_Blood_LMS_100_mius.pl]] **ProbeInfo file: [[Media:whole_blood_LMS_clusters_probeInfo.txt]] **Probe filtering (two layers): ***To eliminate probes that anneal to the low-complexity targets (which would results in non-specific capture and mapping), I checked the ENCODE mappability score for the H1/H2 annealing regions, and remove any probe that have a mappability score of less than 0.6 (an arbitrary cutoff) on one of the two capturing arms. [[Media:check_H1H2_mappability.txt|check_H1H2_mappability.pl]] < whole_blood_LMS_clusters_probeInfo.txt > whole_blood_LMS_clusters_probeInfo_mappability.txt ***Captured regions that have too few CpGs are not informative for haplotype analysis. Therefore I filtered out probes that capture less than 4 CpGs per target. [[Media:cpg_filter.txt|cpg_filter.pl]] < whole_blood_LMS_clusters_probeInfo_uniquely_mapped.txt > whole_blood_LMS_clusters_probeInfo_uniquely_mapped_4cpg-plus.txt **Oligo assembly: [[Media:probe2padlockCpgLib16Jan2014V6.txt|./probe2padlockCpgLib16Jan2014V6.pl]] whole_blood_LMS_clusters_probeInfo_uniquely_mapped_4cpg-plus.txt whole_blood_LMS_clusters_probeInfo_uniquely_mapped_4cpg-plus.txt.seq : 73574 oligos. *Selector probes. **Targets: [[Media:whole_blood_LMS_clusters_BED.txt]]; [[Media:hg19_chr21_unque_input_BED.txt]] **Probe design: ../[[Media:getSelectorProbes.txt|getSelectorProbes.pl]] whole_blood_LMS_clusters_BED.txt "MspI;DpnII,BfaI" > whole_blood_LMR_selectorProbes_MspI_DpnII-BfaI_v2.txt & ../[[Media:getSelectorProbes.txt|getSelectorProbes.pl]] hg19_chr21_unque_input_BED.txt "DpnII,BfaI" > hg19_chr21_unique_DpnII_BfaI_v2.txt **Probe filtering: ***Mappability. ./[[Media:check_selector_H1H2_mappability.txt|check_selector_H1H2_mappability.pl]] < whole_blood_LMR_selectorProbes_MspI_DpnII-BfaI_v2.txt > whole_blood_LMR_selectorProbes_MspI_DpnII-BfaI_v2_mappability.txt ./check_selector_H1H2_mappability.pl < hg19_chr21_unique_DpnII_BfaI_v2.txt> hg19_chr21_unique_DpnII_BfaI_v2_mappability.txt ***Numbers of CpG captured. ./[[Media:selector_cpg_filter.txt|selector_cpg_filter.pl]] < whole_blood_LMR_selectorProbes_MspI_DpnII-BfaI_v2_uniquely_mapped.txt > whole_blood_LMR_selectorProbes_MspI_DpnII-BfaI_v2_uniquely_mapped_3cpg-plus.txt ***Oligo assembly: #Note: I inserted one additional piece of sequence in the linker to make it more flexible. Our standard library-free PCR primers should still work. ./[[Media:probe2padlock2Aug2014V4.txt|probe2padlock2Aug2014V4.pl]] whole_blood_LMR_selectorProbes_MspI_DpnII-BfaI_v2_uniquely_mapped_3cpg-plus.txt ./[[Media:probe2padlock04Aug14_NE.txt|probe2padlock04Aug14_NE.pl]] hg19_chr21_unique_DpnII_BfaI_v2_uniquely_mapped.txt whole_blood_LMR_selectorProbes_MspI_DpnII-BfaI_v2_uniquely_mapped_3cpg-plus.seq: 12,994 hg19_chr21_unique_DpnII_BfaI_v2_uniquely_mapped.txt.seq: 2,539 *Assembly of an oligo pool for ordering. Description # oligos Length Amp. Primers Adaptor Removal Set A: BSPP LMS cluster probes 73,574 148nt V6 USER/DpnII Set B: Selector LMS cluster 12,994 148nt V4 USER/DpnII Set C: Selector chr21 unique 2,539 148nt eMIP_CA_F/R Nt.AlwI/Nb.BrsDI Total: 89,107 *Final oligo pool to order: [[Media:MONOD_v4_04Aug2014.txt.gz]]; [[Media:90k_oligos_04Aug2014.txt.gz]] *Reverse complement linker for Selector probes: GTTGGAGGmCTmCATmCGTTCCTATCCGACGGTAGTGTTTTTTTTTTTAGATGTTATmCGAGGTmCmCGAmC ====Re-design of selector probes==== *I realized that there were a few bugs in the selector probe design script, related to the calculation of chromosome coordinates for the digestion site. As the result, the 3'-ends of those probes are not immediately adjacent to the digestion sites and would not circularized. *I've corrected these errors in a modified script, which reports capture target with 10bp flanking sequences for visual verification. ../[[Media:getSelectorProbes_15Sept14.txt|getSelectorProbes_15Sept14.pl]] whole_blood_LMS_clusters_BED.txt "MspI;DpnII,BfaI" > whole_blood_LMR_selectorProbes_MspI_DpnII-BfaI_v3.txt & ../[[Media:getSelectorProbes_15Sept14.txt|getSelectorProbes_15Sept14.pl]] hg19_chr21_unque_input_BED.txt "DpnII,BfaI" > hg19_chr21_unique_DpnII_BfaI_v3.txt **Probe filtering: ***Mappability. ./[[Media:check_selector_H1H2_mappability.txt|check_selector_H1H2_mappability.pl]] < whole_blood_LMR_selectorProbes_MspI_DpnII-BfaI_v3.txt > whole_blood_LMR_selectorProbes_MspI_DpnII-BfaI_v3_mappability.txt ./check_selector_H1H2_mappability.pl < hg19_chr21_unique_DpnII_BfaI_v3.txt> [[Media:hg19_chr21_unique_DpnII_BfaI_v3_mappability.txt|hg19_chr21_unique_DpnII_BfaI_v3_mappability.txt]] *Sept14 probe set: [[Media:90k_oligos_30Sept2014.txt.gz]] probe set # probes Length Amp primers Oligo assembly script Chris gDNA_MS_v2 8,045 127bp V6(G*T*CATATCGGTCACTGTU//5Phos/GGGTAGTGTGTATCCTG) [[Media:probe2padlock30Sept2014V6.txt|probe2padlock30Sept2014V6.pl]] Kun cancer_hyb_sept14 51,639 110-130bp V8(T*C*TAATCTAGCGCGACGTCU//5Phos/CCACAAGAGGCGCTATG) [[Media:add_PCR_adaptors_Sept2014V8.txt|add_PCR_adaptors_Sept2014V8.pl]] Kun LMS_selector 17,342 124-130bp NE(TGCCTAGGACCGGATCAACT/GCTTCGGTTCACGCAATG) [[Media:probe2padlock30Sept14_NE.txt|probe2padlock30Sept14_NE.pl]] Kun padlock_SNPs 12,974 125bp V4(G*A*CTGGAAGAGCACTGTU//5Phos/AGCCTCATGCGTATCCG) [[Media:probe2padlock29Sept2014V4.txt|probe2padlock29Sept2014V4.pl]] Total 90,000
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