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''' Link to calendar: [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes]] ''' = 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): [[http://genome-tech.ucsd.edu/LabNotes/index.php/Dinh/Dinh_2012/NOTES/2012-3-21]] 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''' <br> '''Note:''' --> = change the labeling to<br> GK0210-001 --> GK0210-002<br> GK0210-002 --> GK0210-001<br> GK0050-005a --> GK0277-002<br> GK0277-002 --> GK0344-004<br> GK0344-004 --> GK0050-005a<br> * 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 results from Binomial test and T-test''' {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''\'''' | align="center" style="background:#f0f0f0;"|'''Binomial test''' | align="center" style="background:#f0f0f0;"|'''T-test''' | align="center" style="background:#f0f0f0;"|'''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 {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Binomial test''' | align="center" style="background:#f0f0f0;"|'''p-value 0.01''' | align="center" style="background:#f0f0f0;"|'''% of ASM''' | align="center" style="background:#f0f0f0;"|'''p-value 0.05''' | align="center" style="background:#f0f0f0;"|'''% 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|| |}<br> ''' Binomial test plotting significant ASM ''' [[File:ASM-Bino_p480E-27-94.png| 300px]] [[File:ASM-Bino_p269E-17-61.png| 270px]] [[File:ASM-Bino_p005-52.png| 310px]] 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 [[File:ASM-Bino_p01-54.png| 300px]] [[File:ASM-Bino_p05-46.png| 270px]] [[File:ASM-Bino_p048-18.png | 290px]] p-value: 0.01 Number of data point: 54 p-value: 0.05 Number of data point: 46 p-value: 0.048 Number of data point: 18 ''' T-Test: plotting significant ASM ''' [[File:ASM-TTest_p153E-51-96.png| 300px]] [[File:ASM-TTest_p005-31.png| 300px]] [[File:ASM-TTest_p002-34.png| 300px]] p-value: 1.53E-51 Number of data point: 96 p-value: 0.005 Number of data point: 31 p-value: 0.002 Number of data point: 34 [[File:ASM-TTest_p0087-86.png| 300px]] [[File:ASM-TTest_p05-55.png| 300px]] p-value: 0.0087 Number of data point: 86 p-value: 0.05 Number of data point: 55 * Continued on 2012_03_23: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-3-23]]
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