Kun:LabNotes/CpgSeq/2011-10-14

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Analysis of BSPP data from Geographic Astrophy (GA) patients

  • 96 blood samples from cases and controls were provided by Kang Zhang's group.
  • Noi performed the BSPP capture and the sequencing was done in BioGem (110929_SN1001)

Summary statistics on sequencing, mapping and CpG methylation calling

Noi should fill in this part.

Regression analysis

  • The working directory is on genome-miner
  /home/kunzhang/mQTL/KangZhang_GA
  • One data set (2200001) was removed due to low capture efficiency and low sequencing depth.
  • Methylation levels from the rest of 95 samples (turned out to be 94) were combined into a matrix (up to 20% missing values was allowed):
  allBED2Matrix.pl BED 76 1 0.1 > KangZhang_GA_BSPP_95_methylationMatrix.txt &
  • I ended up getting a matrix containing 136,417 variable CpG sites.
  • I wrote a Perl Script to perform linear regression on five factors: disease status, sex, age, BMI, smoking: GA_methylation_regression.pl.
  ./GA_methylation_regression.pl  KangZhang_GA_BSPP_95_methylationMatrix.txt > KangZhang_GA_BSPP_95_methylation_regression.txt &
  • Sites that might be significantly contributed by one of these factors were reported.
    • I used a stringent cutoff of 3.5 for the t-statistics on reporting the data in individual files (allowed for more detailed analysis in R).
    • The test statistics for all the tested sites were also reported in a separate file (KangZhang_GA_BSPP_95_methylation_regression.txt).
    • If I simply use a cutoff for |t-value| of 2.5 (equivalent to p=0.014), then I can count how many Cpg sites are significantly correlated with each factor.
     Factor         # sites
     GA             1515 
     Sex            2718 
     Age            2310
     BMI            1861
     Smoking        2002
     GA+Age         249  (somewhat expected as age is a major contributor of GA)
     GA+Sex         53
     GA+BMI         28
     GA+Smoking     34
     It's important to note that no correction for multiple testing was done here. 
     Even with the least stringent FDR correction, no site is significant.