Kun:LabNotes/ExonomeSeq/2009-3-3

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Test whether shaking can improve the circularization[edit]

Padlock capture[edit]

  • Probe sets:
    • Jan09-1: 46,417 probes, 334nM
    • Jan09-2: 53,493 probes, 294nM
    • Jan09-3: 53,828 probes, 186nM
  • Template DNA: PGP1, sheared with Bioruptor, 50ng/ul; 200ug per reaction.
  • Probe:target ratio: 200:1
                       Shaking Control      
   Template(50ng/ul)     4ul    4ul   
   10X AmpLigase buffer  2ul    2ul   
   Jan09-1 (334nM)      3.1ul  3.1ul     
   Jan09-2 (294nM)      4.0ul  4.0ul 
   Jan09-3(unknown)     4.0ul  4.0ul
   H2O                  2.0ul  2.0ul
   95C 2min -> -0.02C/sec to 60C -> (move one tube to ThermoMixer @60C, 400rpm) 
     ->60C 18h 
     -> add 1ul SLN mix (dNTP 5uM, Stoffel 2U/ul, AmpLigase 1U/ul) 
     -> 60C 24h 
     -> 94C 1min -> 37c 1min -> add 2ul Exo I&III
     -> 37C 2h -> 90C 5min -> 4C hold

PCR[edit]

  PCR                           x 2 x 2
  Template:               10ul
  2x iProof master mix:   50ul    
  100uM AmpF6.2          0.4ul    
  100uM AmpR6.2          0.4ul    
  50X SYBG I:            0.4ul    
  H2O                     39ul    
  98C 30sec -> (98C 10sec -> 53C 30sec -> 72C 15sec) x 16 -> 72C 3min -> 4C hold.
  Purified with Qiaquick columns, eluted with 30ul EB.
  Yield:
  PGP1-shaked: 28ng/ul x 30ul
  PGP1-CTRL:   24ng/ul x 30ul

2nd round PCR with Solexa adaptor[edit]

  PCR                           x 2 
  Template:                1ul
  2x iProof master mix:   50ul    
  100uM AmpF6.2Sol       0.4ul    
  100uM AmpR6.2Sol       0.4ul    
  H2O                     49ul    
  98C 30sec -> (98C 10sec -> 56C 30sec -> 72C 15sec) x 5 -> 72C 3min -> 4C hold.
  Purified with Qiaquick columns, eluted with 30ul EB.
  Yield:
  PGP1-shaked-LONG: 33ng/ul x 30ul
  PGP1-CTRL-LONG: 34ng/ul x 30ul

Ship six libraries to VCU for end-sequencing[edit]

  1. PGP1AC: 278nM x 15ul
  2. PGP1AC-Norm1: 157nM x 15ul
  3. PGP1AC-Norm2: 121nM x 15ul
  4. PGP1S: 167nM x 15ul
  5. PGP1D: 273nM x 15ul
  6. PGP1CTR: 172nM x 15ul

Data analysis[edit]

  • The first four libraries were sequenced on 03/07/09.
  Library        #raw reads  #mappable reads  #targets
  PGP1AC         8,948,696   147,867/152,555  7,593,058
  PGP1AC-Norm1   9,933,208   143,790/152,555  8,016,337
  PGP1AC-Norm2   5,008,442   139,647/152,555  4,143,134
  PGP1S          7,974,771   145,500/152,555  6,592,144
  Representation bias
  File:2009-03-07-Exome-Jan09 set-representation-bias.png
  • Summary
    • The four libraries have quite similar bias, and the distribution of bias is quite similar to the hybrid capture method published in the Nature Biotech paper.
    • Post-capturing normalization didn't seem to work. Three possibilities:
      • The ds-DNA nuclease lost its activity;
      • The complexity of the library is >10x higher than the Oct08 set.
      • The library was not biased enough to be normalized.

Perhaps it takes longer for the DNA to anneal.

Histogram of target counts[edit]

File:2009-03-07-Exome-Jan09-PGP1AC-dist.png File:2009-03-07-Exome-Jan09-PGP1S-dist.png
File:2009-03-07-Exome-Jan09-PGP1AC-norm1-dist.png File:2009-03-07-Exome-Jan09-PGP1AC-norm2-dist.png