Kun:LabNotes/ExonomeSeq/2010-1-22

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Test the N2 adaptor protocol for library construction[edit]

  • In this protocol, captured PCR amplicons are digested with Mme I. The digested DNA fragments carry a 2bp overhang at the 3'-ends, and a phosphate group at the 5'-end. Such fragments can be ligated with a Y-shape adaptor with a degenerate 2bp overhang at the 5'-end, amplified with the standard Illumina PCR primers, and sequenced.
  • In this experiment, I'll test this protocol with one exome library (CV-iB-AB from Alice) and one BSPP library.

1st round PCR[edit]

  • I need to amplify enough DNA for Mme I digestion.
                                  x 2
    Template                 1ul
    2X iProof MasterMix    100ul
    100uM AmpF6.3            1ul
    100uM AmpR6.3            1ul
    50X SYBG I             1.6ul
    98C 30sec -> (98C 10sec -> 60C 20sec -> 72C 10 sec) x 8 -> 72C 2min
  • Purified with Qiaquick columns.
  • Measured with Nanodrop:
    • CV-iPS-B: 116ng/ul x 30ul
    • BSPP-B2: 112ng/ul x 30ul

Mme I digestion[edit]

  • Calculation of Mme I:
    • One unit of Mme I can digest 1ug of phiX 174 DNA.
    • phiX 174 DNA contain 5 Mme I cutting sites in 5389bp.
    • One captured PCR amplicon contain 2 Mme I cutting sites in ~250bp.
    • To cut 1ug of the PCR amplicon, we need 2/250/(5/5389)=8.6 units of Mme I.
   DNA              10ul
   10X NEBuffer 4    4ul
   Mme I             4ul
   1mM SAM           3ul (I use 75uM since the tube of SAM has been used before)
   H2O              29ul
   37C 1h -> Purified with Qiaquick columns
    • Measure concentration with Nanodrop:
      • CV-iPS-B: 23ng/ul x 30ul
      • BSPP-B2: 26ng/ul x 30ul
  File:2010-01-22-MmeI-digestion.png

Ligation[edit]

  • Prepare the adaptor:
    100uM PE_N2_adaptor    20ul
    100uM PE_b_A           20ul
    10x Stoffel buffer     10ul
    H2O                    50ul
    94C 2min -> 0.2C/sec to 20C -> 4C hold.
  • Set up the ligation reaction. Assuming one ligation site per 100bp dsDNA, adaptor:target=20:1
   Digested DNA (~200ng)  10ul
   2X QuickLigase buffer  25ul
   20uM annealed adaptors  6ul
   QuickLigase           2.5ul
   H2O                     7ul
   RT for 15min -> Purified with the MinElute columns, eluted with 15ul EB.

PCR[edit]

                                   x 2
    Template                 10ul      
    2x iProof mix           100ul       
    Solexa_PCR_up (10uM)    0.8ul        
    Solexa_PCR_lo (10uM)    0.8ul        
    H2O                      89ul     
    50X SYBG I              0.8ul     
    98C 30sec -> 8 cycles of (98C 10sec -> 62C 20sec -> 70C 15 sec) -> 72C 2min
    Add additional cycles:  4 cycles of (98C 10sec -> 70C 15 sec) -> 72C 2min
 File:2010-01-26-ligation-PCR.png  File:2010-01-26-ligation-PCR-real-time-curves.png

Size selection and quantification[edit]

 File:2010-01-27-size-selection.png  File:2010-01-28-library-quant.png
 
 Gel quantification:
 Library                   conc(ng/ul)       conc(nM)
 KZ-Exome-CViB-N2-Jan28     5.2ng/ul           31nM
 KZ-BSPP-B2-N2-Jan28        2.1ng/ul           10nM
 KZ-DigiQ-FS-300p-Jan27     0.6ng/ul          2.8nM
 KZ-DigiQ-FS-600p-Jan27     1.3ng/ul          6.0nM

Conclusion[edit]

  • Library construction seems to be successful. There are clean smears at the expected size range.
  • Ideally, the libraries should be constructed from the first-round captured PCR amplicons.
  • I could have used only 1/3 of the DNA for Mme I digestion, since only 1/3 of the digested DNA was enough for the ligation. In other words, this protocol need only ~300ng of captured PCR amplicons.
  • Two additional PCR cycles are enough.
  • I need to do PAGE size selection on the ligation-PCR products. The captured PCR amplicons don't need to be size selected.