Kun:LabNotes/CpgSeq/2008-3-18: Difference between revisions

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   Sbjct  327  GGATGGGGAGGG  316
   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

Revision as of 07:06, 29 March 2008

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. 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