Kun:LabNotes/CpgSeq/2008-3-18
Exp. 1 Cpg capture with a new batch of bisulfite treated Jurkat DNA from 03/17/08
Sigma x2 Zymo x 2 Template 7 3 10X buffer(fresh) 1 1 Cpg28k probe(218nM) 2 2 H2O 0 4 Tubes: A: Sigma, dNTP 10nM B: Sigma, dNTP 50uM C: Zymo, dNTP 10nM D: Zyno, dNTP 50uM
Exon capture control
GM20431 131ng/ul 7 (10/12/2006) 10X buffer(fresh) 1 E55kv1 probe(220nM) 2 (01/12/2008) H2O 0
94C 10min -> 60C 10min -> add 1ul NSL mix (Stoffel 2U/ul, Ampligase 0.5U/ul, dNTP 10nM or 50uM) -> 60C 1h -> 45 cycles of (94C 1min -> 60C 1h) -> 60C hold -> 37C 1min -> add 2ul Exo I&III, 37C 2h -> 94C 5min -> 4C hold.
PCR
Template 12ul 2X iProof Mastermix 100ul 100uM AmpF6.2 0.8ul 100uM AmpR6.2 0.8ul 50X SYBG I 0.8ul H2O 85ul Split into two tubes. 98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 15 -> 72C 5 min -> 15C hold.
File:2008-03-21-Cpg-E55kv1-captured-PCR.png A: Sigma, dNTP 10nM B: Sigma, dNTP 50uM C: Zymo, dNTP 10nM D: Zymo, dNTP 50uM E: E55kv1, dNTP 10nM F: E55kv1, dNTP 50uM
To test whether some of the high MW bands are concatemers, I did Alu I digestion on the MinElute purified amplicons. 8ul DNA + 1ul 10X NEBuffer2 + 1ul Alu I -> 37C 30min -> 75C 10min. File:2008-03-21-capured-amplicon-AluI-cut.png
PAGE purification of amplicon D, F, and Cosmic C(03/17/08)
File:2008-03-21-PAGE.png Resuspended the pellets in 15ul ddH2O.
Blunt end cloning
PAGE purified amplicons: 3.5ul 10X ligation buffer: 0.5ul Zero Blunt vector: 0.5ul T4 DNA ligase: 0.5ul 16C 1h -> 4C overnight.
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
- Clean sequences after trimming off vectors
- Blast comparison to the probes
- Summary of reads
- Probe information files:
Reamplify the PAGE size selected Zymo, dNTP 50uM for Solexa sequencing (04/10/08)
Template 5ul 10X Platinium Taq buffer 20ul 50mM MgCl2 6ul 100uM AmpF6.2SolH 0.8ul 100uM AmpR6.2SolH 0.8ul 10mM dNTP 4ul 50X SYBG I 0.8ul H2O 163ul iTaq(5U/ul) 4ul 94C 2min -> (94C 30S -> 58C 30sec -> 72C 20S) x 6 -> 72C 3min -> 15C hold.