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]
- PGP1AC: 278nM x 15ul
- PGP1AC-Norm1: 157nM x 15ul
- PGP1AC-Norm2: 121nM x 15ul
- PGP1S: 167nM x 15ul
- PGP1D: 273nM x 15ul
- 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 |