Arichard:Notebook/midas2014Aug28: Difference between revisions

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>Andrew
>Andrew
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| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''Sample'''
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| 1||No headspace||9||Headspace, Pol I
| 1||No headspace, no Pol I||9||No Pol I
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| 2||No headspace||10||Headspace, Pol I
| 2||No headspace, no Pol I||10||No Pol I
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| 3||No headspace||11||Headspace, Pol I
| 3||No headspace, no Pol I||11||No Pol I
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| 4||No headspace||12||Headspace, no Pol I
| 4||No headspace, no Pol I||12||Normal
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| 5||No headspace, no Pol I||13||Headspace, no Pol I
| 5||No headspace||13||Normal
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| 6||No headspace, no Pol I||14||Headspace, no Pol I
| 6||No headspace||14||Normal
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| 7||No headspace, no Pol I||15||NTC from array 2
| 7||No headspace||15||Array 2 NTC
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| 8||No headspace, no Pol I||16||Tube NTC
| 8||No headspace||16||Tube NTC
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Revision as of 19:13, 11 September 2014

MIDAS run, August 28, 2014

Motivation for different conditions

  • We have observed a fraction of samples, between 1/4 and 1/2 in some experiments, that display "over-amplification" as determined by wide histograms for bin coverage and large numbers of CNV calls, many of which we can assume to be spurious. Our hypothesis is that the 20 ul headspace above the microwells (23 ul total volume - 3 ul combined well volumes = 20 ul) provides a space for reactions to "run-away".
  • We have also seen evidence form Eric's device that the Pol I step is not necessary for Nextera. Furthermore, I don't think this condition was fully explored when Jeff and I originally settled on this method as our standard protocol.

Tests

2 hypotheses to test, in combination --> 4 total conditions: (1) No headspace, no Pol I; (2) No headspace; (3) No Pol I: (4) Normal protocol

  • No headspace: Remove the headspace after adding master mix by pipeting from the edge of each array. Phi 29 should have approx. 72 second diffusion time for a distance of 100 um. Add oil overlay as usual.
  • No Pol I: After extracting MDA amplicons from microwells into 1 ul H2O, do no denature with ALS. Instead, add 6 ul H2O and pipet up and down 5-10 times before adding 2 ul 5X Nextera Buffer and 1 ul 1:50 Nextera. Proceed as usual.

Samples

  • NeuN positive cortex from patient 703 sorted May 20, 2014
Array loading ' ' '
No headspace No headspace Normal Normal
No headspace No headspace Normal Normal
Tube Sample Tube Sample
1 No headspace, no Pol I 9 No Pol I
2 No headspace, no Pol I 10 No Pol I
3 No headspace, no Pol I 11 No Pol I
4 No headspace, no Pol I 12 Normal
5 No headspace 13 Normal
6 No headspace 14 Normal
7 No headspace 15 Array 2 NTC
8 No headspace 16 Tube NTC