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:
77/94 are on-target capture, 16 are primer concatemers, 1 seems to be cloning artifact. File:Cpg031808-dist.png
Reamplify the PAGE size selected Zymo, dNTP 50uM for Solexa sequencing (04/10/08)
Template 5ul 10X Platinium Taq buffer 40ul 50mM MgCl2 12ul 100uM AmpF6.2SolH 1.6ul 100uM AmpR6.2SolH 1.6ul 10mM dNTP 8ul 50X SYBG I 1.6ul H2O 326ul iTaq(5U/ul) 8ul split into 4x100ul 94C 2min -> (94C 30S -> 58C 30sec -> 72C 20S) x 9 -> 72C 3min -> 15C hold. Mix all 4x100ul amplicon, purified 100ul with Qiaquick, 100ul with Zymo Concentrator-25. Qiaquick: 22ng/ul x 30ul Zymo: 28ng/ul x 15ul Clonewell size selection on the Qiauick purified DNA. Purify the remaining 200ul amplicon with Qiaquick, 34ng/ul x 30ul
Make shotgun sequencing library
Mme I digestion
Qiaquick purified DNA 26ul x 34ng/ul 10X NEBuffer 4: 6ul 1mM SAM 2ul Mme I (2U/ul) 5ul H2O 21ul 37C 90min -> 80C 20min -> 4C hold. I ran the digestion in 6% PAGE gel without purification, it turned out that the DNA migration pattern was totally off, probably due to DNA:protein interaction.
Perform another PCR on the remaining PAGE size selected DNA (04/10/08)
Template 5ul 2x Taq Master mix 200 100uM AmpF6.2SolH 1.6ul 100uM AmpR6.2SolH 1.6ul 1mM dUTP 4ul 50X SYBG I 3.2ul H2O 172ul split into 4x100ul 94C 2min -> (94C 30S -> 58C 30sec -> 72C 20S) x 8 -> 72C 3min -> 15C hold. Mix all 4x100ul amplicon, purified with Zymo Concentrator-25.
Make shotgun sequencing library
Mme I digestion
Qiaquick purified DNA 26ul x 34ng/ul 10X NEBuffer 4: 6ul 1mM SAM 2ul Mme I (2U/ul) 5ul H2O 21ul 37C 90min -> 85C 20min -> 4C hold. MinElute purification -> 6% TBE PAGE size selection -> the DNA migration pattern is still not right I think it is due to the 85C head inactivation step, which denatured DNA. So what I was see are ssDNA or partially annealed ds/ssDNA. File:2008-04-11-Cpg-Jurkat-2nd-PCR-MmeI-cut.png
Perform another PCR on the remaining PCR amplicon (27ng/ul)
Template 4ul 2x Taq Master mix 150ul 100uM AmpF6.2SolH 1.2ul 100uM AmpR6.2SolH 1.2ul 1mM dUTP 3ul 50X SYBG I 2.4ul H2O 129ul split into 3x100ul 94C 2min -> (94C 30S -> 58C 30sec -> 72C 20S) x 6 -> 72C 3min -> 15C hold. Purified with Zymo Concentrator-25.
Make shotgun sequencing library
Mme I digestion
DNA 20ul x 29ng/ul 10X NEBuffer 4: 4ul 1mM SAM 1.3ul Mme I (2U/ul) 4ul H2O 10ul 37C 90min 4C hold. Qiaquick purification -> 6% TBE PAGE size selection The DNA migration pattern was still not right. Looks like Mme I didn't make any cut at all. Repeat the digestion by adding 36ul H2O, 5ul 1mM SAM, 4ul Mme I, 5ul 10X NEBuffer 4. 37C 4h. File:2008-04-13-Cpg-Jurkat-MmeI-Size-selection.png
End repair (Epicentre End-it repair kit)
Resuspended the pallet in 30ul ddH2O, add 3ul USER enzyme, 37C 4h. Add 5ul 10x Buffer, 5ul dNTP mix, 5ul ATP, 1ul Enzyme mix. RT overnight. Purified with MinDlute kit. 16ng/ul x 10ul
Blunt-end ligation
25ng/ul end-repaired DNA 10ul 100uM Solexa_1 pair 0.5ul 100uM Solexa_2 pair 0.5ul 2X Quick ligase buffer 15ul Water 3ul Quick Ligase (NEB) 1ul RT 10min -> Purified with Qiaquick column. PAGE size selection, ethanol precipitation
Nick-translation
DNA 15ul 10X ThermoPol Buffer 2ul 10mM dNTP 0.4ul 1mg/ml BSA 2ul 8U/ul Bst pol 1ul 65C 25min -> place on ice
PCR
Nick-translated DNA 10ul 2X iProof master mix 100ul 100uM Solexa_PCR_up 1ul 100uM Solexa_PCR_lo 1ul H2O 87.3ul 50X SYBG I 0.8ul 98C 30sec -> 10 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold
Perform another round of PCR on the 04/10/08 amplicon (27ng/ul)
Template 5ul 10X Platinium Taq buffer 40ul 50mM MgCl2 12ul 100uM AmpF6.2SolH 1.6ul 100uM AmpR6.2SolH 1.6ul 10mM dNTP 8ul 1mM dUTP 2ul 50X SYBG I 1.6ul H2O 326ul JumpStart Taq(5U/ul) 4ul split into 4x100ul 94C 2min -> (94C 30S -> 58C 30sec -> 72C 20S) x 6 -> 72C 3min -> 15C hold. Purified 4X100ul amplicons with 2x Zymo DNA concentrator-25 columns. Eluted DNA from each column with 30ul EB buffer. Yield: 47ng/ul x 60ul. File:2008-04-12-Cpg-Jurkat-3rd-PCR-amplicons.png
Mme I digestion
DNA 30ul x 47ng/ul 10X NEBuffer 4: 5ul 1mM SAM 2.5ul Mme I (2U/ul) 5ul H2O 10ul 37C 1h. Qiaquick purification -> 6% TBE PAGE size selection