Dinh/Dinh 2012/NOTES/2012-3-21

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Sequence dependent test on HAPMAP data

  • Working directory (genome-miner): /home/nplongth/Noi_scratch/ASM_HAPMAP1362-1454_2012_02_03/DD_SequenceDependentTest
  • Discussed this with Noi and Dr. Zhang
Sequence dependent means that there is a consistent preference of methylation on one allele versus another across multiple individuals.
  • Dr. Zhang suggested Binomial test
  • Considerations for the binomial test:
1. Using all data from *cpg.txt file generated from Robert's pipeline (ignoring p-value)
2. Ignore data where the two alleles have exactly the same methylation fraction (no preference)
3. Identify the favored allele in each individual, and increment count for the favored allele at a SNP:CpG set.
4. Do not consider sites where there are more than 2 different favored alleles
5. Let x = the count for the least favored allele
6. Use Math::CDF qw(pbinom) with x, n = number of data, p = probability of preference on allele = 0.5
   Null hypothesis is that the sample prefer allele 1 and allele 2 both with probability 0.5
   Reject null if the p-value is low (more samples prefer one allele over the other)
7. Accept null if p value is greater than 0.05 (5%)
8. Resulted in 117 SNP:CPG (Sequence dependent ASM)
  • Command:
dinh@genome-miner:/home/nplongth/Noi_scratch/ASM_HAPMAP1362-1454_2012_02_03$ DD_SequenceDependentTest/asmBinomialTest.pl MFASM.QValues-cpg.list.txt > DD_SequenceDependentTest/binomialtest.SequenceDependent
  • Noi suggested the T-test
  • Considerations for the t-test:
1. Using all data from *cpg.txt file generated from Robert's pipeline (ignoring p-value)
2. Get the difference in methylation level between the two alleles, making sure that the difference is always relative to the same allele.
3. Require minimum 5 data points
4. Calculate the t-statistic: (mean difference)/SEM (use Statistics::Descriptive)
   Null hypothesis is that the average of the differences is 0.
   Reject null if the p-value is low (the average of the differences is not 0)
5. Calculate the p-value using Math::CDF qw(pt) with t-stat, df = number of data - 1, and centered at 0.
6. Accept null if p value is greater than 0.05 (5%)
7. Resulted in 1244 SNP:CPG (Sequence dependent ASM) 
  • Command:
dinh@genome-miner:/home/nplongth/Noi_scratch/ASM_HAPMAP1362-1454_2012_02_03$ DD_SequenceDependentTest/asmTTest.pl MFASM.QValues-cpg.list.txt > DD_SequenceDependentTest/ttest.SequenceDependent

Double check Binomial test with OpenOffice Math

10:127584276:127584265 3:2796414:2796339 17:21220301:21220322
0.271|0.591 0.786|0.417 0.865|0.000
0.228|0.629 0.562|0.468 0.733|0.000
0.296|0.439 0.906|0.825 0.816|0.027
NA 0.850|0.040 0.455|0.000
0.267|0.613 0.632|0.340 0.600|0.000
0.427|0.513 0.644|0.246 0.125|0.146
0.178|0.534 0.661|0.769 0.688|0.000
0.436|0.548 0.475|0.452 0.784|0.013
0.302|0.706 NA NA
0.145|0.681 0.532|0.529 1.000|0.074
0.411|0.403 NA 0.194|0.000
0.214|0.695 0.842|0.577 0.827|0.012
p=0.005859375 p=0.0107421875 p=0.005859375
  • First and last SNP:CPG have n=10 and k=1 (n is number of data and k is the least favored allele count)
' n 11 10
k 1 1
P(k=0) 0 0.0004882813 0.0009765625
P(k=1) 1 0.0053710938 0.009765625
SUM --- 0.005859375 0.0107421875
  • probabilities were calculated using: =(FACT(n)/(FACT(k)*FACT(n-k)))*0.5^k*(0.5)^(n-k)

Double check Ttest with OpenOffice Math

4:7319795:7319781 14:95874397:95874396 13:38445196:38445195
NA NA 0.000|0.714
0.000|0.303 0.000|0.952 0.000|0.700
NA NA NA
0.750|0.975 0.009|0.981 0.000|0.933
NA 0.005|0.950 0.048|0.867
0.292|0.851 NA 0.000|0.970
NA 0.000|0.947 0.000|0.361
NA NA NA
NA NA NA
0.718|0.930 NA NA
0.571|1.000 0.000|0.977 NA
NA NA NA
p=0.039493696 p=1.71390437762721e-07 p=0.009553314
  • Calculate the differences then t-statistics and pvalue
' 4:7319795:7319781 14:95874397:95874396 13:38445196:38445195
diff 0.303 0.952 0.714
diff 0.225 0.972 0.7
diff 0.559 0.945 0.933
diff 0.212 0.947 0.819
diff 0.429 0.977 0.97
diff 0.361
mean 0.3456 0.9586 0.7495
SEM 0.1472270356 0.0148425065 0.2198933833
T-stat 2.3473949515 64.584778741 3.4084699999
P-value 0.0393695698 1.72149891022281E-007 0.0095374811