MIDAS run, August 28, 2014[edit]
Motivation for different conditions[edit]
- 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.
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[edit]
- 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
|
|
- The Pol I condition samples had no signal on the gels after Nextera PCR, so I didn't size select them (samples 5-8, 12-14, and 16).
- It looks like the no headspace, no Pol I condition worked, so I am going to use this method for the next experiment.
- I won't know for sure if the no headspace condition is better than with headspace until I sequence them, but the bright band for sample 11 (with headspace) looks like over-amplification, so I am encouraged.
File:Midas140828GelsSlide1.jpg
File:Midas140828GelsSlide2.jpg
File:Midas140828GelsSlide3.jpg
File:Midas140828GelsSlide4.jpg
File:Midas140828GelsSlide5.jpg
File:Midas140828GelsSlide6.jpg
Sequencing[edit]
- I got confused about the order in which I was adding barcode adapters for samples 9, 10, and 11, but I was consistent.
- There are only two possibilities for each, and they do not overlap with any other submitted libraries.
- These libraries were submitted for sequencing without screening to save time.
- They will be sequenced in HL173.
Tube #
|
SeqID
|
Index
|
Barcode
|
2 |
AR51-PX703May20-Aug28-2 |
2 |
CGATGT
|
4 |
AR52-PX703May20-Aug28-5 |
4 |
ACAGTG
|
9 |
AR53-PX703May20-Aug28-6or11 |
6 or 11 |
GCCAAT or GGCTAC
|
10 |
AR54-PX703May20-Aug28-7or12 |
7 or 12 |
CAGATC or CTTGTA
|
11 |
AR55-PX703May20-Aug28-8or13 |
8 or 13 |
ACTTGA or CCATGA
|
|