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
- Performed binomial test using asmBinomialTest.pl script written by Dinh: Media:asmBinomialTest.txt
./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
- Performed t-test using asmTTest.pl script written by Dinh: Media:asmTTest.txt
./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_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
./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
- 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 | 4,005 | 648 | XX |
SNP on CpG ASM | 703 | 7,633 | XX |
Total | 4,708 | 8,281 | XX |
p-value 0.05 | |||
SNP-free ASM | 7,658 | 995 | 162 |
SNP on CpG ASM | 852 | 8,128 | 460 |
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_PVal0.05.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 | 4,005 | 85.07% | 7,658 | 89.99% |
SNP on CpG | 703 | 14.93% | 852 | 10.01% |
Total | 4,708 | 8,510 | ||
T-test | ||||
SNP-free ASM | 648 | 7.83% | 995 | 10.91% |
SNP on CpG | 7,633 | 92.17% | 8,128 | 89.09% |
Total | 8,281 | 9,123 |
Binomial test plotting significant ASM
File:ASM-Bino p480E-27-94.png File:ASM-Bino p269E-17-61.png File:ASM-Bino p005-52.png File:ASM-Bino p01-54.png p-value: 4.80E-27 Number of data point: 94 p-value: 2.69E-17 Number of data point: 61 p-value: 0.005 Number of data point: 52 p-value: 0.01 Number of data point: 54
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
- Continued on 2012_03_23: [[3]]