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  • Data filters at what stage of NGS data analysis?

    Greetings friends!

    I seek help with data that I have : 3 time points, 3 genotypes, 3 replicates for each of these = 27 libraries

    The goal is to find genes that have different time expression profiles amongst 2 or more genotypes.

    After our 1st round of data analysis, (including TMM normalization), the time course graphs and box plots were so noisy in terms of high std error at each time point, that it was hard to say if expression profile of one genotype was overlapping or distinct from that for the other genotypes! R code attached at bottom of this post.

    So in short - we now need to employ data filters to check and reduce noise in our data. Some ideas are
    removing genes that have low expression (count) levels
    removing genes that have high variance across replicates
    removing genes that have low variance across time (constitutively expressed genes are biologically less interesting)

    So my question to you is what stage of my analysis do I employ these filters?
    On the raw data itself, prior to normalization?
    Or should I perform the TMM normalization, use the norm factors to transform my data to non-integer normalized counts and then filter (in which case I think I cannot fit them into negative binomial model, right?)

    count = read.table("Input.txt", sep="\t", header=T)                     					
    #$#$ read in raw count mapped data
    f.count = count[apply(count[,-c(1,ncol(count))],1,sum) > 27,]                               
    #$#$ filter ou genes with total read count < 27 across all libraries
    f.dat = f.count[,-c(1,ncol(count))]                                                         
    #$#$ select only read count, not rest of data frame
    S = factor(rep(c("gen1","gen2","gen3"),rep(9,3)))                                           
    #$#$ define group
    Time = factor(rep(rep(c("0","10","20"),rep(3,3)),3))         								
    #$#$ define time
    Time.rep = rep(1:3,9)                                                                        
    #$#$ define replicate
    Group = paste(S,Time,Time.rep,sep="_")                                                         
    #$#$ define group_time_replicate
    #$#$ load edgeR package
    f.factor = data.frame(files = names(f.dat), S = S , Time = Time, lib.size = c(apply(f.dat,2,sum)),norm.factors = calcNormFactors(as.matrix(f.dat)))  
    #$#$  make data for edgeR method
    count.d = new("DGEList", list(samples = f.factor, counts = as.matrix(f.dat)))               
    #$#$  make data for edgeR method
    design = model.matrix(~ Time + S)                                                           
    #$#$  make design data for edgeR method
    count.d = calcNormFactors(count.d)                                                          
    #$#$  Normalize TMM
    glmfit.d = glmFit(count.d, design, dispersion = 0.1)                                        
    #$#$  Fit the Negative Binomial Gen Lin Models
    lrt.count = glmLRT(count.d, glmfit.d)                                                       
    #$#$  Likelihood ratio tests
    result.count = data.frame(f.count, lrt.count$table)                                         
    #$#$  combining raw data and results from edgeR
    result.count$FDR = p.adjust(result.count$p.value,method="BH")                               
    #$#$  calculating the False Discovery Rate
    write.table(result.count, "edgeR.Medicago_count_WT_Mu3.txt",sep="\t",row.names=F)           
    #$#$  saving the combined data set

  • #2
    See this:


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