ignore/code/analysis/mousehybrid/seqdepth2ngenes.R

In edsgard/scphaser: Phase Variants Within Genes Using Allele Counts

###Assess complexity under varying parameters
###NB: The paths are relative to the root of the scphaser git repo
###
###SYNOPSIS
###library('devtools')
###devtools::load_all()
###source("ignore/code/analysis/mousehybrid/seqdepth2ngenes.R")


##Params
sys = 'rs13'
sys = 'dna'


##*###
##Dirs
##*###

if(sys == 'dna'){
    cloud.dir = '/mnt/kauffman/edsgard/cloud/btsync/work/rspd'
}
if(sys == 'rs13'){
    cloud.dir = '/Volumes/Data/cloud/btsync/work/rspd'
}
prefilt_rds_dir = '../nogit/data/mousehybrid/all_snps/downsampled'

##OUT
out_rds_dir = '../nogit/data/mousehybrid'
out_pdf_dir = './ignore/res/mousehybrid/perf/pdf'


##*###
##Files
##*###

##OUT
param2ngenes_rds = file.path(out_rds_dir, 'seqdepth2ngenes.rds')

##Libs
source('./ignore/code/analysis/performance.R')
library('BiocParallel')
library('dplyr')
library('tidyr')

main <- function(){

    ##read data
    load('../nogit/data/mousehybrid/mousehybrid.rda')

    ##create acset
    featdata = mousehybrid$featdata
    ref = featdata[, 'c57']
    alt = featdata[, 'cast']
    featdata = cbind(featdata, ref, alt, stringsAsFactors = FALSE)
    acset = new_acset(featdata, mousehybrid$refcount, mousehybrid$altcount, mousehybrid$phenodata)
    lapply(acset, dim) #167,443 x 336
    length(unique(acset[['featdata']][, 'feat'])) #11,590
        
    ##Filter vars with 0 counts
    acset = filter_zerorow(acset)
    lapply(acset, dim) #167,443
    length(unique(acset[['featdata']][, 'feat'])) #11,590

    ##Call gt
    min_acount = 3
    fc = 3 #75/25
    acset = call_gt(acset, min_acount, fc)

    ##Filter variants on number of cells where ASE towards the same allele (as to remove probable false positive calls)
    alpha = 0.1
    mono.ase = 0.1
    if(!(mono.ase == 0)){
        acset = filter_homovars(acset, alpha = alpha, mono.ase = mono.ase)
    }
    lapply(acset, dim) #104,933 x 336

    
    ##*###
    ##Randomly sample n counts
    ##*###
    altcount = acset[['altcount']]
    refcount = acset[['refcount']]
    totcount = altcount + refcount

    ##number of reads
    ntot = sum(totcount)
    ntot #139,192,303
    ncount = c(seq(25e6, 125e6, 25e6), ntot)
    
    ##permutation iterations
    npermiter = 10 #10
    perm_iter = 1:npermiter
    
    ##specify paramset as all possible combinations of the params
    paramset = expand.grid(perm_iter, ncount, stringsAsFactors = FALSE)
    colnames(paramset) = c('perm_iter', 'ncount')
    
    ##parallelization
    ncores = 80
    bp_param = BiocParallel::MulticoreParam(workers = ncores)

    ##Filter vars and feats
    nparamset = nrow(paramset)
    acset_list = BiocParallel::bplapply(1:nparamset, filter_acset_par, BPPARAM = bp_param, paramset = paramset, acset = acset)
    ##status: fin (11.30 -> 11.40, 80 cores)

    ##get number of genes
    ngenes = unlist(lapply(acset_list, function(j.acset){length(unique(j.acset[['featdata']][, 'feat']))}))
    param2ngenes = cbind(paramset, ngenes)

    ##get number of variants
    nvars = unlist(lapply(acset_list, function(j.acset){nrow(j.acset[['featdata']])}))
    param2ngenes = cbind(param2ngenes, nvars)

    ##get fraction of genes
    n.bg.genes = 20268 ##see log.sh
    frac.genes = param2ngenes[, 'ngenes'] / n.bg.genes
    param2ngenes = cbind(param2ngenes, frac.genes)

    ##add col mean number of reads per cell
    n.cells = ncol(altcount)
    print(n.cells)
    ncount.percell = param2ngenes[, 'ncount'] / n.cells
    param2ngenes = cbind(param2ngenes, ncount.percell)
    
    ##Dump
    saveRDS(param2ngenes, file = param2ngenes_rds)
    ##status: fin

    summary(param2ngenes[, 'ngenes'])

    
    ##*###
    ##Plot
    ##*###
    param2ngenes = readRDS(param2ngenes_rds)

    library('ggplot2')

    errbar.w = 2.5e6 / 336
    
    gg = ggplot(param2ngenes, aes_string(x = 'ncount.percell', y = 'ngenes'))
    gg = gg + geom_line(stat = 'summary', fun.y = 'mean')
    gg = gg + geom_errorbar(stat = 'summary', fun.data = mean_se, width = errbar.w) #width = errbar.w

    ##tick breaks
    ##gg = gg + coord_cartesian(ylim = c(800, 3050))
    ##gg = gg + scale_y_continuous(breaks = seq(1000, 3000, 500))
    ##gg = gg + scale_x_continuous(breaks = c(seq(25e6, 125e6, 25e6)))
    
    ##Background
    gg = gg + theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank(), axis.line = element_line(colour = "black")) + theme(panel.background = element_blank())
    gg = gg + theme(axis.text = element_text(colour="black", size = 15), axis.ticks = element_line(colour = 'black'), axis.title = element_text(size = 15))
    gg = gg + xlab('Mean number of allele counts per cell')
    gg = gg + ylab('Number of phasable genes')
    gg = gg + theme(axis.line.x = element_line(color="black", size = 0.5), axis.line.y = element_line(color="black", size = 0.5))

    pdf.h = 5
    pdf.w = 5
    
    j.pdf = file.path(out_pdf_dir, 'nallelecount2ngenes.pdf')
    dir.create(dirname(j.pdf), recursive = TRUE)
    pdf(j.pdf, height = pdf.h, width = pdf.w)
    plot(gg)
    dev.off()


    gg = ggplot(param2ngenes, aes_string(x = 'ncount.percell', y = 'frac.genes'))
    gg = gg + geom_line(stat = 'summary', fun.y = 'mean')

    ##tick breaks
    ##gg = gg + coord_cartesian(ylim = c(800, 3050))
    ##gg = gg + scale_y_continuous(breaks = seq(1000, 3000, 500))
    ##gg = gg + scale_x_continuous(breaks = c(seq(50, 336, 50), 336))
    
    ##Background
    gg = gg + theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank(), axis.line = element_line(colour = "black")) + theme(panel.background = element_blank())
    gg = gg + theme(axis.text = element_text(colour="black", size = 15), axis.ticks = element_line(colour = 'black'), axis.title = element_text(size = 15))
    gg = gg + xlab('Mean number of allele counts per cell')
    gg = gg + ylab('Number of phasable genes')
    gg = gg + theme(axis.line.x = element_line(color="black", size = 0.5), axis.line.y = element_line(color="black", size = 0.5))

    pdf.h = 5
    pdf.w = 5

    j.pdf = file.path(out_pdf_dir, 'nallelecount2fracgenes.pdf')
    dir.create(dirname(j.pdf), recursive = TRUE)
    pdf(j.pdf, height = pdf.h, width = pdf.w)
    plot(gg)
    dev.off()

}

filter_acset_par <- function(j.param, paramset, acset, nmincells = 5, nminvar = 2){

    j_ncount = paramset[j.param, 'ncount']
    
    altcount = acset[['altcount']]
    refcount = acset[['refcount']]
    totcount = altcount + refcount
    ncount = sum(totcount)

    frac.down = j_ncount / ncount

    ##keep relative frequency btw alt and ref (skip sampling in this step)
    alt.frac = sum(altcount) / ncount
    ref.frac = 1 - alt.frac
    alt.frac.down = alt.frac * frac.down
    ref.frac.down = ref.frac * frac.down
    
    ##downsample
    altcount.down = downsample.matrix(altcount, alt.frac.down)
    refcount.down = downsample.matrix(refcount, ref.frac.down)
    
    ##set to downsampled data
    acset[['refcount']] = refcount.down
    acset[['altcount']] = altcount.down

    ##Call gt
    min_acount = 3
    fc = 3 #75/25
    acset = call_gt(acset, min_acount, fc)
    
    ##filter vars and genes
    acset_filt = filter_acset(acset, nmincells, nminvar)

    return(acset_filt)
}

downsample.matrix <- function(count.mat, frac.down, pseudo.count = 0){

    ##keep relative frequency between cols (skip sampling in this step)
    col2ncount = apply(count.mat, 2, sum)
    col2ncount.down = ceiling(col2ncount * frac.down)

    ##add pseudocount (skip, since we are down-sampling there is no need to do this)
    count.mat = count.mat + pseudo.count
    col2ncount = apply(count.mat, 2, sum)

    ##proportion of each element in a col
    probs.bycol = t(count.mat) / col2ncount
    ##each row: probs for a col

    ##for each col downsample    
    ##count.down = apply(Hmisc::rMultinom(probs.bycol, col2ncount.down), 1, table) ##Hmisc::rMultinom does not accept different sizes ("m = col2ncount.down").
    ##rownames(count.down) = rownames(count.mat)
    ##colnames(count.down) = colnames(count.mat)
    
    count.down = matrix(nrow = nrow(count.mat), ncol = ncol(count.mat), dimnames = list(rownames(count.mat), colnames(count.mat)))
    n.cols = length(col2ncount)
    for(j.col in 1:n.cols){
        jcol.ncount.down = col2ncount.down[j.col]
        jcol.probs = probs.bycol[j.col, ]
        jcol.count.down = rmultinom(1, jcol.ncount.down, prob = jcol.probs)
        count.down[, j.col] = jcol.count.down
    }

    return(count.down)
}