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

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==Reamplify the PAGE size selected Zymo, dNTP 50uM for Solexa sequencing (04/10/08)==
==Reamplify the PAGE size selected Zymo, dNTP 50uM for Solexa sequencing (04/10/08)==
     Template                    5ul
     Template                    5ul
     10X Platinium Taq buffer  20ul
     10X Platinium Taq buffer  40ul
     50mM MgCl2                 6ul
     50mM MgCl2                 12ul
     100uM AmpF6.2SolH        0.8ul
     100uM AmpF6.2SolH        1.6ul
     100uM AmpR6.2SolH        0.8ul
     100uM AmpR6.2SolH        1.6ul
     10mM dNTP                  4ul
     10mM dNTP                  8ul
     50X SYBG I                0.8ul
     50X SYBG I                1.6ul
     H2O                      163ul
     H2O                      326ul
     iTaq(5U/ul)                4ul
     iTaq(5U/ul)                8ul
     94C 2min -> (94C 30S -> 58C 30sec -> 72C 20S) x 6 -> 72C 3min -> 15C hold.
    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.

Revision as of 01:08, 11 April 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, 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

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.