Kun:LabNotes/Haplotyping/2009-2-12
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Comparison of MDA using N6, LN6 and N9 primers[edit]
- The objective of this experiment is to compare the amplification bias of several variants of MDA protocols:
- Control: N6 primer: 30C
- LN6 (LNA primer): 30C, 37C, 30C + trehalose
- N9: 30C + trehalose
- The template DNA is GM20431, 30pg/reaction (5 copies of diploid genome)
- Two replicates of GM20431 and NTC (20 reactions in total)
Prepare Alkaline Lysis Solution (ALS) and Neutralization Solution (NS)[edit]
- ALS: (400mM KOH, 100mM DTT, 10mM EDTA)
- Prepare 5M KOH: weigh one KOH pallet, add nuclease free H2O to make 5M KOH. Make fresh solution every week.
- Mix 8ul 5M KOH, 10ul 1M DTT, 102ul nuclease free H2O.
- NS buffer (0.4M HCl, 0.6M Tris.HCl pH 7.5):
Set up MDA reactions[edit]
- Perform serial dilution of GM20431 gDNA to 30pg/ul.
- Perform UV treatment of ALS and NS buffer 10ul/tube for 5 min;
- Prepare 2X SYBR Green I solution from the 50X stock solution;
- Mix 1ul template with 1.5ul ALS buffer, RT for 3min;
- Transfer to cool block;
- Add 1.5ul NS buffer;
- Add 16ul Master mix:
x13 x9 Tre(-) Tre(+) Trehalose(1.2M) 0ul 5.0ul 10x RepliPhi phi-29 buffer 2.0ul 2.0ul 1mM primer 1.0ul 1.0ul 25mM dNTP 0.8ul 0.8ul 2X SYBR Green I 1.0ul 1.0ul H2O 9.7ul 4.7ul RepliPhi Phi-29 (100U/ul) 1.0ul 1.0ul
- Incubate at 30C for 10h on Opticon4, take one fluorescent read every 6 minutes.
File:2009-02-12-MDA-N6.png File:2009-02-12-MDA-LN6.png File:2009-02-12-MDA-LN6-Tre.png File:2009-02-12-MDA-N9-Tre.png File:2009-02-12-MDA-LN6-37C.png
Illumina Infinium Data[edit]
GM20431 | Correct Calls | LOH | Incorrect Calls | No Calls |
LN6.GType | 186950 | 56700 | 56735 | 249883 |
N6.GType | 230134 | 51403 | 45395 | 223336 |
N9+Tre.GType | 335118 | 54811 | 48774 | 111565 |
30pg.GType | 303840 | 52613 | 47240 | 146575 |
LN6+Tre.GType | 299575 | 56480 | 50434 | 143779 |
Relative intensity compared with 200ng gDNA
Amplicon | GM20431 N6.GType | GM20431 30 pg.GType | GM20431 LN6.GType | GM20431 LN6+Tre.GType | GM18507 200 ng.GType | GM18507 30pg.GType | GM20431 N9+Tre.GType |
STDEV | 1.069 | 0.975 | 1.391 | 0.904 | 0.208 | 0.889 | 0.835 |
Mean | -0.326 | -0.124 | -0.336 | -0.214 | 0.018 | -0.487 | -0.160 |
It looks like the amplicon using the N9 primer + Trehalose has the most number of correct calls.