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==Colony PCR & sequencing== I pick 16 colonies from the CpG plate, 8 from the Cosmic plate and 8 from the E55kv1 plate. PCR: 2X TaqMaster mix 15ul 10uM M13 F/R primer mix 0.6ul H2O 14.4ul 94C 2min -> 35 cycles of (94C 30sec -> 52C 30sec -> 72C 30sec) -> 72C 3min Check with Argarose gel, 4ul amplicons per lane, the ladder is Invitrogen Low Mass ladder (4ul). Cpg #1: Cpg A1 Cosmic #1: Cosmic A1 Cpg #2: Cpg B1 Cosmic #2: Cosmic B1 Cpg #3: Cpg C1 Cosmic #3: Cosmic C1 Cpg #4: Cpg D1 Cosmic #4: Cosmic D1 Cpg #5: Cpg E1 E55kv1 #1: E55kv1 A1 Cpg #6: Cpg F1 E55kv1 #1: E55kv1 A1 Cpg #7: Cpg H1 E55kv1 #1: E55kv1 D1 Cpg #8: Cpg B2 E55kv1 #1: E55kv1 E1 ===Analysis of sequencing results=== ====Cosmic amplicons==== Insert(incl. H1&H2) Target Note Cosmic #1: 235bp FRMD4A exon 1 mismatch in H2 Cosmic #2: 65bp, contains AP1V6 and AP2V2 sequences, seems to due to chimerism in probe prep Cosmic #3: 226bp RIMS2 exon 100% match Cosmic #4: Similar to #2, also has amplification adaptors Raw sequences of #2 >Cosmic2-T7.ab1 XXXXXXXXXXGCAGCTGAATAGGAACGATGAGCCTCCAACACCCAATCTT AAAGTGATTGCTCCATGTTTAACAGTGCTCTTCCAGTCTACAACAGTGCT CTTCCAGTCTACCTAGCCTCATGCGTATCCGATCCAGAAGGCCAGAAATG GCAGTGTCGGACCTCGATAACATCTGCAGCTGAATAGGAACGATGAGCCT CCAACACCCAATCTTAAAGTGATTGCTCCATGTTTAACAGTGCTCTTCCA GTCTACAACAGTGCTCTTCCAGTCTACCTAGCCTCATGCGTATCCGATCC AGAAGGCCAGAAATGGCAGTGTCGGACCTCGATAACATCTGCA Raw sequences of #4 >Cosmic4-T7.ab1 XXXXXXXXXXXXTGCAGATGTTATCGAGGTCCGACAAACCAATTGCACAG CACACGATCGGATACGCATGAGGCTATGTAGACTGGAAGAGCACTGTTAC ACTAAGAGCCCACTGCCAGAGGTTATGGAGTTGGAGGCTCATCGTTCCTA TTCAGCTGCAGATGTATCGAGGTCCGACAAACCAATTGCACAGCACACGA TCGGATACGCATGAGGCTATGTAGACTGGAAGAGCACTGTTACACTAAGA GCCCACTGCCAGAGGTTATGGAGTTGGAGGCTCATCGTTCCTATTCAGCT GC ====Cpg #1==== The capturing sequences were mapped to the probe targeting the CpG island at chr22:38387887-38388790. The insert is 192bp, same as the expected size. I did blast pair-wise alignment between the insert and the targeted genomic sequence. Query 34 CGTCGTTTTAGTTGGAATAGTTTTAAGTATAAGTCGTCGTCGGTGGAGTATGAGTTTTTG 93 ||||| | || ||||| || | ||| | ||| || |||||| |||| |||||| || Sbjct 187 CGTCGCTCCAGCTGGAACAGCCTCAAGCACAAGCCGCCGTCGGCGGAGCATGAGTCCCTG 246 Query 94 TTTTTTGAGGAGCGCGGCGGCGGCGTTCGGGTTTGCGAGGTTGTCGCGGACGAGGGGTCG 153 | | || ||||||||||||||||| ||||| |||||||||| ||||||||||||| || Sbjct 247 CTCTCTGCGGAGCGCGGCGGCGGCGCCCGGGTCTGCGAGGTTGCCGCGGACGAGGGGCCG 306 Query 154 TCGTGGGTCGTATTTTTGTATATTTTATACGTTTATTACGTTTATTATGGGTTTTATTTG 213 || ||| || | || | | | ||| | || || || | ||| || || Sbjct 307 CCGCGGGCCGCACCCCTGCACACCCCACACGCCCACCACATTCATCACGGGCCCCATCTG 366 Query 214 GTGTATCGTTATCGTTATTATCGTCGGACGTTGTTTTTCGATAATAGGGATTTGGTGGAT 273 | | | || | || | | || |||||| ||| |||| || ||||| | |||||| Sbjct 367 GCGCACCGCCACCGCCACCACCGCCGGACGCTGTCCCTCGACAACAGGGACTCGGTGGAC 426 Query 274 TTGGTCG 280 ||| || Sbjct 427 CTGGCCG 433 ====Cpg #2==== The capturing sequences were mapped to the probe targeting the CpG island at chr20:61,136,225-61,137,000. The insert is 182bp, same as the expected size. I did blast pair-wise alignment between the insert and the targeted genomic sequence. Query 41 GGTGGTTTGGGTCGGTGTTTAGTTTAGGGTTTCGCGGTTAGAGTTAATTATGGCGCGTTT 100 ||||| |||||||||| | || ||||| ||||| |||| || |||||||| | Sbjct 70 GGTGGCCTGGGTCGGTGCTCAGCCCAGGGTCCCGCGGCCAGAGCCAACCATGGCGCGCCT 129 Query 101 GGTTTTTTTTAGATAAACGCGCGTTTACGTTTTTGTTGGGTTTTGGAATTAGTTTAATAT 160 ||| | | ||||||||||||| |||| | || |||| ||||||||||| | |||| Sbjct 130 GGTCTCCTCCAGATAAACGCGCGCCCACGTCTCTGCTGGGCTTTGGAATTAGCTCAATAC 189 Query 161 TCGTTTAGACGTAGAGTGCGTGTGTTTTGTTAGGATAGACGTTATTGTTGTGTGGTGTTT 220 || || ||||| |||||||||||| || |||| |||||| |||| |||| |||| || Sbjct 190 TCATTCAGACGCAGAGTGCGTGTGCCCTGCCAGGACAGACGTCATTGCTGTGCGGTGCTT 249 Query 221 GAGTGTTATTAATAAGTTATAGAAGAGAAATTCGGAAATAAGGGA 265 ||||| || |||||| | ||||||||||| |||||| |||||| Sbjct 250 GAGTGCCATCAATAAGCCACAGAAGAGAAATCCGGAAACAAGGGA 294 ====Cpg #3==== Tandem concatemers of AP1V4 and AP2V2, plus half of the probes for c20:19945840w. I think this is due to the chimerism of the first PCR step. ====Cpg #4==== Similar to Cpg #3: tandem concatemers of AP1V4 and AP2V2, plus half of the probes for c20:30048155c. I think this is due to the chimerism of the first PCR step. ====Cpg #5==== The first 22-235bp are AT rich sequence that I haven't been able to mapped to the genome. 236-295bp can be mapped to multiple positions in hr19:42463268-42490100, which contains mostly SST1 satellite repeats. I think this is due to probe design issues. ====Cpg #6==== The capturing arms were mapped to the probe targeting c22:38387974w. Insert is 172bp, which is what I expected. However, I can't map the insert to the target, which is probably due to problem with C:T mismatches. I did the alignment manually, which turned out to be successful. The reason Blast didn't pick up the alignment was due to sequencing errors in homopolymer runs. ====Cpg #7==== The capturing sequences were mapped to the probe targeting the CpG island at chr19:41485375-41485793. The insert is 211bp, same as the expected size. I did blast pair-wise alignment between the insert and the targeted genomic sequence. Query 44 AGGAAGGTTAGTTTTTGAGAGATAGATATGTTTGTTTTATTTTTTTGTCGGTTTTTTTTT 103 ||||||| || ||||||||| ||| ||| ||| || ||| ||| || || | Sbjct 567 AGGAAGGCCAGCCTTTGAGAGACAGACATGCTTGCCCCATCCCCTTGCCGGCTTCCTTCT 508 Query 104 TCGTTTTTGCGTCGAGTTGTGGTTATATTTTTCGATGAGGGTAGAGGGCGATAGGCGTGA 163 ||| ||||||||| ||||| || |||| |||||||||| ||||||||| |||||||| Sbjct 507 CCGTCCCTGCGTCGAGCTGTGGCTACATTTCTCGATGAGGGCAGAGGGCGACAGGCGTGA 448 Query 164 TAATTATTTTTTTGAAGTTTTGCGGGTATTTTTTTGCGGGTAGACGATGAGCGTTTGGGA 223 || | || |||||| | |||||| | | ||||||| ||||||||||| ||||| Sbjct 447 CAACCACCTTCTTGAAGCTCTGCGGGCACCCTCCTGCGGGTGGACGATGAGCGCCTGGGA 388 Query 224 GGTCGTTGTTTTTGGTTGGGGAGCGTTCGTTTGGATTTAGTTTAGTAAAGAGGTTGTTTC 283 || |||||| |||||||||||||| |||| ||||| || ||| ||||||| || | | Sbjct 387 GGCCGTTGTCCTTGGTTGGGGAGCGCTCGTCTGGATCCAGCCTAGCAAAGAGGCTGCTCC 328 Query 284 GGATGGGGAGGG 295 |||||||||||| Sbjct 327 GGATGGGGAGGG 316 ====Cpg #8==== The capturing sequences were mapped to the probe targeting the CpG island at c19:42463450c. I did blast pair-wise alignment between the insert and the targeted genomic sequence. Query 51 TGGGTCGTGTGGTCGGTTGGTAGTCGTCGTTTCGGCGGTTTTAGTTTGGGGGTTTGTGAA 110 |||||||||||| ||||||| ||||||||| | || ||| ||||||||||| |||||| Sbjct 429 TGGGTCGTGTGGCCGGTTGGCAGTCGTCGTCCCAGCAGTTCCAGTTTGGGGGTCTGTGAA 370 Query 111 GGTTTGGGTAACGTGGGTATGGGCGTCGGATTCGTA-GGGTTTTTATTTTTTTTTTATTC 169 || |||| |||||||| || ||||||| | || | ||||||| || | || | Sbjct 369 GGCCTGGGCAACGTGGGCATCGGCGTCGAACCCGCAGGGGTTTTCATCCCCTCCCCATCC 310 Query 170 GGAGTAGTTTTTTTGTTAGGTTGGATTTAGACGAGCGTTTTTTAATTAAGGATAACGGTT 229 |||| || | |||| |||| ||||| ||||||||| | || ||||| ||||| Sbjct 309 GGAGCAGCCTCTTTGCTAGGCTGGATCCAGACGAGCGCTCCCCAACCAAGGACAACGGCC 250 Query 230 TTTTAGGCGTTTATCGTTTATTCGTAGGAGGGTGTTCGTAGAGTTTTAAGAAGGTGGTTG 289 | ||||| | || || | || |||| |||| || |||| || ||||||||||||| Sbjct 249 TCCCAGGCGCTCATTGTCCACCCGCAGGAAGGTGCCCGCAGAGCTTCAAGAAGGTGGTTG 190 Query 290 TTACG 294 | ||| Sbjct 189 TCACG 185 ====Summary of Cpg capture==== *5/8 amplicons were expected targets. *2/8 contains mostly primer concatemers due to issues in probe prep. *1/8 could not be mapped uniquely because the probe was design for a repetitive region not masked in UCSC genome draft. ===I submitted another 96 clones to Agencourt for sequencing, received the data on 04/08/08=== *[[Media:cpg031808.fa.screen.trimmed.txt|Clean sequences after trimming off vectors]] *[[Media:cpg031808.fa.screen.trimmed.blastn.CpgMIP28k.out.txt|Blast comparison to the probes]] *[[Media:Cpg031808-summary.xls|Summary of reads]] *Probe information files: **[[Media:cpgIslandExt_250bp_1N_chr6.probes.xls|Chr6 probes]] **[[Media:cpgIslandExt_250bp_1N_chr19.probes.xls|Chr19 probes]] **[[Media:cpgIslandExt_250bp_1N_chr20.probes.xls|Chr20 probes]] **[[Media:cpgIslandExt_250bp_1N_chr21.probes.xls|Chr21 probes]] **[[Media:cpgIslandExt_250bp_1N_chr22.probes.xls|Chr22 probes]] 77/94 are on-target capture, 16 are primer concatemers, 1 seems to be cloning artifact. [[Image:Cpg031808-dist.png|400px]]
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