Noi/NOTES/2012-3-21: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Noi
No edit summary
>Noi
No edit summary
 
(56 intermediate revisions by the same user not shown)
Line 1: Line 1:
''' Link to calendar: [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes]] '''
= ASM analysis of UCLA SZ data set =
= 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 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]]
* 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
  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]]

Latest revision as of 18:39, 25 March 2012

Link to calendar: [[1]]

ASM analysis of UCLA SZ data set[edit]

  • 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_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
\' 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  
 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 File:ASM-Bino p05-46.png  File:ASM-Bino p048-18.png
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 File:ASM-TTest p005-31.png  File:ASM-TTest p002-34.png   
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 File:ASM-TTest p05-55.png
p-value: 0.0087   Number of data point: 86        p-value: 0.05   Number of data point: 55    
  • Continued on 2012_03_23: [[3]]