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Rui:DNAseq analysis on Hiseq120313
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===Validation=== * multiple bands / unknown sequence in PCR results -> confirm primers on gDNA * negative SNCs -> positive aliquot PCR ====PCR primers==== {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''PCR-ID''' | align="center" style="background:#f0f0f0;"|'''Gene''' | align="center" style="background:#f0f0f0;"|'''Cat.''' | align="center" style="background:#f0f0f0;"|'''chr''' | align="center" style="background:#f0f0f0;"|'''pos''' | align="center" style="background:#f0f0f0;"|'''refBase''' | align="center" style="background:#f0f0f0;"|'''B2B3(GT)''' | align="center" style="background:#f0f0f0;"|'''positive aliquot''' | align="center" style="background:#f0f0f0;"|'''Pf''' | align="center" style="background:#f0f0f0;"|'''Pr''' | align="center" style="background:#f0f0f0;"|'''length''' | align="center" style="background:#f0f0f0;"|'''position''' |- | SNC1||GLT25D2 ||exon||1||183907978||G||G=7/T=3||B2_Indx80,B2_Indx93,B3_Indx93|| CAGGGAGCCTTCATAGCTCA|| ACCTGAGTGACACGGAGACC||189||>chr1:183907885+183908073 |- | SNC2||FLNB ||exon||3||58104657||C||C=6/G=3||B2_Indx80,B2_Indx82,B3_Indx92|| TTTGGTACCCAAAGGTAAACTGA|| CAACCCATCTGCTCTCCATT||191||>chr3:58104557+58104747 |- | SNC3||PPP1R9A ||exon||7||94918006||G||A=3/G=6||B3_Indx82,B3_Indx95,B3_Indx96|| AGTGTGCAGCAGGTTTCTCA|| CCTCTCAGATATGCAGAATGCTC||223||>chr7:94917911+94918133 |- | SNC4||POU5F1P3 (in CLEC4A)||exon, pseudogene||12||8286643||C||C=13/G=6||B2_Indx81,B2_Indx83,B2_Indx87,B2_Indx93,B3_Indx83,B3_Indx88||GAGACCCAACAGCCTCAAAA||AGTGAGAGGCAACCTGGAGA||191||>chr12:8286540+8286730 |- | SNC5-6||POU5F1P4 (in ASH1L)||exon, pseudogene||1||155403537||G||C=5/G=6||B2_Indx81,B2_Indx88,B2_Indx91,B2_Indx93,B3_Indx74||AACAATTTGCCAAGCTCCTG||TGCAAGAGGGTTTCTGCTTT||bad||chr1; chr8; chr6 (X5) |- | ||POU5F1P4 (in ASH1L)||exon, pseudogene||1||155403505||G||A=5/G=5||B2_Indx88,B2_Indx91,B2_Indx93,B3_Indx74,B3_Indx95|||||||| |- | SNC7||(in DAB1)||Ensemble psedogene||1||58694451||A||A=12/G=3||B2_Indx77,B2_Indx86,B3_Indx92|| CTGGGGTTCTTGGACCTTCT|| GATGCTCTGGGCCTCTGTC||178||>chr1:58694376+58694553 |- | SNC9||||Ensemble psedogene||9||7598174||C||C=11/T=4||B2_Indx73,B2_Indx81,B2_Indx84,B3_Indx73|| CACTGGCAGCAAGTCAATCT|| GCCATCCAACCACTCAGTCT||158||>chr9:7598095+7598252 158bp |- | SNC10||(in PLXNC1)||intron; repeats||12||94620564||T||C=3/T=13||B2_Indx91,B3_Indx75,B3_Indx96|| CCAGGTAAAGTGACATTTTTGG|| AGGACCAGGCAGTCACAAAC||369||>chr12:94620480+94620848 369bp |- | SNC11-12||||Ensemble psedogene||9||7598190||A||A=10/G=5||B2_Indx73,B2_Indx79,B2_Indx81,B2_Indx84,B3_Indx73||GGGTTCCTGCTTTCACAAAA||TTGCTGAAGACCACATGCTT||247||>chr9:7598026+7598272 247bp |- | ||||Ensemble psedogene||9||7598203||C||C=12/T=5||B2_Indx73,B2_Indx79,B2_Indx81,B2_Indx84,B3_Indx73|||||||| |- | SNC14||GOT2L2||exon, pseudogene||10||93897290||A||A=11/G=3||B2_Indx74,B2_Indx86,B3_Indx84|| CCAAAAGCTCAACAGCAACA|| TCCACTCCTCATGTTCCACA||176||>chr10:93897208+93897383 176bp |- | SNC13||EIF4A1P8 (in CPEB3)||exon, pseudogene||1||173111263||C||C=7/T=3||B3_Indx82,B3_Indx84,B3_Indx92||TCACCCAGGGTTGGTTCTAC||TGGGAGTCACTGAAGCCTTT||204||>chr1:173111163+173111366 204bp |- | SNC15||(in EP300)||intron||22||41572232||G||A=3/G=8||B2_Indx79,B2_Indx88,B2_Indx91|| GAGGATCAGGATCAACAATGG|| GTGCTCAGCCTGCTGTACCT||388||>chr22:41571936-41572323 388bp |- | SNC16||UPF3A||exon||13||115047559||C||C=5/T=3||B2_Indx74,B2_Indx78,B2_Indx86|| GTTCCGCCCTCTCTTCCTC|| CCTTTTGAGCTCCTTGTCCA||204||>chr13:115047437-115047640 204bp |- | SNC377||SEZ6 ||exon; repeats||17||27296926||T||C=3/T=10||B2_Indx74,B2_Indx91,B3_Indx76|| AGGCAGCAGGAAGAAGTCTG|| CAGCAGGAAAGACTGGTTGG||151||>chr17:27296875-27297025 151bp |} * PCR product from primers on P30 gDNA [[File:4.27.12_PCR1-7.jpg|400px]] [[File:4.27.12_PCR8-14.jpg|400px]] {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''PCR-ID''' | align="center" style="background:#f0f0f0;"|'''length''' | align="center" style="background:#f0f0f0;"|'''PCR result''' |- | SNC1||189||good |- | SNC2||191||weak |- | SNC3||223||good |- | SNC4||191||3 bands |- | SNC5-6||bad||3 bands |- | |||| |- | SNC7||178||good |- | SNC9||158||good |- | SNC10||369||good |- | SNC11-12||247||weak+smear |- | |||| |- | SNC14||176||good |- | SNC13||204||good |- | SNC15||388||no |- | SNC16||204||>10 bands |- | SNC377||151||good |} ====Sequence results==== {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''PCR-ID''' | align="center" style="background:#f0f0f0;"|'''length''' | align="center" style="background:#f0f0f0;"|'''PCR result''' | align="center" style="background:#f0f0f0;"|'''Gene''' | align="center" style="background:#f0f0f0;"|'''chr''' | align="center" style="background:#f0f0f0;"|'''pos''' | align="center" style="background:#f0f0f0;"|'''refBase''' | align="center" style="background:#f0f0f0;"|'''B2B3(GT)''' | align="center" style="background:#f0f0f0;"|'''Fib''' | align="center" style="background:#f0f0f0;"|'''P30''' | align="center" style="background:#f0f0f0;"|'''B2''' | align="center" style="background:#f0f0f0;"|'''B3''' | align="center" style="background:#f0f0f0;"|'''B2_MDA''' | align="center" style="background:#f0f0f0;"|'''B3_MDA''' | align="center" style="background:#f0f0f0;"|'''Notes''' |- | SNC1||189||good||GLT25D2 ||1||183907978||G||G=7/T=3||G||G||G||G||G (T? C?)||N/A||confirm w/ aliquot |- | SNC2||191||weak||FLNB ||3||58104657||C||C=6/G=3||SNC1? T?||C [330bp]||C||SNC1? T?||no match||SNC1? N/A||repeat PCR (probmatic) |- | SNC3||223||good||PPP1R9A ||7||94918006||G||A=3/G=6||G||G [270bp]||G||G||G||chr1|| |- | SNC4||191||3 bands||POU5F1P3 (in CLEC4A)||12||8286643||C||C=13/G=6||C/G||G/A/C||G||A||||||C: chr12; G:chr8/1 |- | SNC5-6||bad||3 bands||POU5F1P4 (in ASH1L)||1||155403537||G||C=5/G=6|||||||||||||| |- | ||||||POU5F1P4 (in ASH1L)||1||155403505||G||A=5/G=5|||||||||||||| |- | SNC7||178||good||(in DAB1)||1||58694451||A||A=12/G=3||A||A||A||A||A||A||clear peak, confirm w/ aliquot |- | SNC9||158||good||||9||7598174||C||C=11/T=4||off target||C/T||chr21?||chr12?||chr2?||chr2?||repeat PCR (probmatic) |- | SNC10||369||good||(in PLXNC1); repeat||12||94620564||T||C=3/T=13||||||||||||||repeats |- | SNC11-12||247||weak+smear||||9||7598190||A||A=10/G=5|||||||||||||| |- | ||||||||9||7598203||C||C=12/T=5|||||||||||||| |- | SNC14||176||good||GOT2L2||10||93897290||A||A=11/G=3||A||A||A||A||A||A||clear peak, confirm w/ aliquot |- | SNC13||204||good||EIF4A1P8 (in CPEB3)||1||173111263||C||C=7/T=3||C||C||C||C||C/T/G||C||chr17,12,13 etc.; mosaic |- | SNC15||388||no||(in EP300)||22||41572232||G||A=3/G=8||G||SNC13?||G||G||G/A||no match||3 B2 aliquot |- | SNC16||204||>10 bands||UPF3A||13||115047559||C||C=5/T=3||chr. 12?-off||chr. 17?-off||chr. 17: T||chr. 17?||||||chr13-C; chr17-C, T |- | SNC377||151||good||SEZ6; repeat||17||27296926||T||C=3/T=10||T||T||mhits, T||off target||chr.15?||off target||repeats |}
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