Noi/NOTES/2012-3-21

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Link to calendar: [[1]]

ASM analysis of UCLA SZ data set

  • After discussing with Dr. Zhang and Dinh about how to identify sequence-dependent ASM, Dr. Zhang suggested to identify the consistency of sequence preference DNA methylation across individual
  • After having more discussion with Dinh, she helped me out to write scripts for sequence-dependent ASM using binomial test and t-test by testing on ASM HAPMAP data (total 12 samples): [[2]]
on genome-miner: /home/nplongth/Noi_scratch/ASM_HAPMAP1362-1454_2012_02_03/DD_SequenceDependentTest
  • I did the same analysis on UCLA SZ data set
on genome-miner: /home/nplongth/Noi_scratch/ASM_UCLA/combined_ASM_UCLA-4batches_2012_01_24
  • Since there were mislabeling of some samples at the beginning from UCLA and after mapping and ASM analysis, and I generated many files. I still kept all labeling the same as original, but made the correction when generated the methylation matrix or other sample_list files for any analysis. Here are the list of samples used in analysis and correction.

Correct data mislabeling in UCLA.SZ data set
Note: --> = change the labeling to
GK0210-001 --> GK0210-002
GK0210-002 --> GK0210-001
GK0050-005a --> GK0277-002
GK0277-002 --> GK0344-004
GK0344-004 --> GK0050-005a

  • Generated the list of MFASMAnalysisQValues.GK0XXXXXX.cpg.txt in UCLA_MFASM_Qcpg_list.txt: Media:UCLA_MFASM_Qcpg_list.txt
  • Note: For these two tests (binomial and t-test), we require minimum 5 data points and p-value of 0.05

Binomial test

./asmBinomialTest.pl UCLA_MFASM_Qcpg_list.txt > UCLA_SZ_SeqDepTest_Binomial_PVal0.05.txt
  • I got total 8,510 SNP:CpG sites showing significant ASM using p-value cutoff 0.05. However, when I looked closer to the methylation level on each allele showing significant ASM by this test, it showed that some sites contain very close methylation level or have the methylation fraction 0 on both alleles. I may try to increasing stringency by lowering the p-value and observe the resulting methylation level on each allele.
awk '{if ($98 <=0.01) print $0;}' UCLA_SZ_SeqDepTest_Binomial_PVal0.05.txt > UCLA_SZ_SeqDepTest_Binomial_PVal0.01.txt
  • After lowering p-value cutoff, I got total 4,708 SNP:CpG sites.
  • Note: In the first column of the table, 13:38445196:38445195 --> chromosome position: SNP position: CpG position

T-test

./asmTTest.pl UCLA_MFASM_Qcpg_list.txt > UCLA_SZ_SeqDepTest_TTest_PVal0.05.txt
  • I got total 9,123 SNP:CpG sites showing significant ASM, p-value 0.05.
awk '{if ($98 <=0.01) print $0;}' UCLA_SZ_SeqDepTest_TTest_PVal0.05.txt > UCLA_SZ_SeqDepTest_TTest_PVal0.01.txt
  • I got 8,281 SNP:CpG sites, p-value 0.01
    • --> explain more
  • Extract number of significant ASM
    • SNP-free ASM:
    • SNP on CpG ASM:
./getnoSNPCpG.pl UCLA_SZ_SeqDepTest_Binomial_PVal0.01.txt > UCLA_SZ_SeqDep_Binomial-noSNP_PVal0.01.txt 
./getSNPonCpG.pl UCLA_SZ_SeqDepTest_Binomial_PVal0.01.txt > UCLA_SZ_SeqDep_Binomial-SNPonCpG_PVal0.01.txt 
./getnoSNPCpG.pl UCLA_SZ_SeqDepTest_Binomial_PVal0.05.txt > UCLA_SZ_SeqDep_Binomial-noSNP_PVal0.05.txt 
./getSNPonCpG.pl UCLA_SZ_SeqDepTest_Binomial_PVal0.05.txt > UCLA_SZ_SeqDep_Binomial-SNPonCpG_PVal0.05.txt 
./getnoSNPCpG.pl UCLA_SZ_SeqDepTest_TTest_PVal0.01.txt > UCLA_SZ_SeqDep_TTest-noSNP_PVal0.01.txt 
./getSNPonCpG.pl UCLA_SZ_SeqDepTest_TTest_PVal0.01.txt > UCLA_SZ_SeqDep_TTest-SNPonCpG_PVal0.01.txt 
./getnoSNPCpG.pl UCLA_SZ_SeqDepTest_TTest_PVal0.05.txt > UCLA_SZ_SeqDep_TTest-noSNP_PVal0.05.txt 
./getSNPonCpG.pl UCLA_SZ_SeqDepTest_TTest_PVal0.05.txt > UCLA_SZ_SeqDep_TTest-SNPonCpG_PVal0.05.txt 
  • Comparing the the results from Binomial test and T-test
' Binomial test T-test overlapped Binomial/T-test
p-value 0.01
SNP-free ASM 3,898 639 38
SNP on CpG ASM 810 7,642 366
Total 4,708 8,281 404
p-value 0.05
SNP-free ASM 7,502 978 156
SNP on CpG ASM 1,008 8,145 466
Total 8,510 9,123 622
  • Form these numbers it suggested that there were less than 10% of the significant ASM were consistent by the two tests. Moreover, from the table below, more than 80% of significant ASM from binomial test belong to SNP-free ASM. On the opposite, more than 80% of ASM by t-test belong to SNP on CpG. I need to observe closer and compare which test suitable to identify significant ASM appropriately.
./getnoSNPCpG.pl overlapped_UCLA_SZ_SeqDep_BinoTTest_PVal0.01.txt > overlapped_UCLA_SZ_SeqDep_BinoTTest-noSNP_PVal0.01.txt
./getSNPonCpG.pl overlapped_UCLA_SZ_SeqDep_BinoTTest_PVal0.01.txt > overlapped_UCLA_SZ_SeqDep_BinoTTest-SNPonCpG_PVal0.01.txt
./getnoSNPCpG.pl overlapped_UCLA_SZ_SeqDep_BinoTTest_PVal0.05.txt > overlapped_UCLA_SZ_SeqDep_BinoTTest-noSNP_PVal0.05.txt
./getSNPonCpG.pl overlapped_UCLA_SZ_SeqDep_BinoTTest_PVal0.05.txt > overlapped_UCLA_SZ_SeqDep_BinoTTest-SNPonCpG_PVal0.05.txt
  • --> describe more
Binomial test p-value 0.01 % of ASM p-value 0.05 % of ASM
SNP-free ASM 3,898 82.80 7,502 88.16%
SNP on CpG 810 17.20 1,008 11.84%
Total 4,708 8,510
T-test
SNP-free ASM 639 7.72 978 10.72%
SNP on CpG 7,642 92.28 8,145 89.28%
Total 8,281 9,123


Binomial test plotting significant ASM

  • SNP-free ASM examples:

File:UCLA ASM BinoT-2.png File:UCLA ASM BinoT-3.png File:UCLA ASM BinoT-4.png

 p-value: 4.80E-27 Number of data point: 94      p-value: 3.72E-26    Number of data point: 91         p-value: 0.0099    Number of data point: 54                               
  • SNP on CpG ASM example:

T-Test: plotting significant ASM

  • SNP-free ASM examples

File:UCLA ASM TTest-1.png File:UCLA ASM TTest-2.png

p-value: 1.07E-43    Number of data point: 45                        p-value: 2.45E-36   Number of data point: 39
  • SNP on CpG ASM example:

File:UCLA ASM-SNPonCpG TTest-1.png

p-value: 1.53E-51    Number of data point: 96