tests/testthat/test-norm.R

In LTLA/scuttle: Single-Cell RNA-Seq Analysis Utilities

# Tests for normalisation methods
# library(scuttle); library(testthat); source("setup.R"); source("test-norm.R")

ncells <- ncol(sce)
ngenes <- nrow(sce)
dummy <- counts(sce)

# Making up some size factors.
X <- sce
ref <- runif(ncells)
sizeFactors(X) <- ref

test_that("normalizeCounts works as expected", {
    out <- normalizeCounts(dummy, ref, center_size_factors=FALSE)
    expect_equal(out, log2(t(t(dummy)/ref)+1))

    # With size factor centering.
    sf <- ref/mean(ref)
    out <- normalizeCounts(dummy, ref)
    expect_equal(out, log2(t(t(dummy)/sf)+1))

    # Without log-transformation.
    out <- normalizeCounts(dummy, ref, log=FALSE)
    expect_equal(out, t(t(dummy)/sf))

    # With asinh transforms.
    out <- normalizeCounts(dummy, ref, transform="asinh")
    expect_equal(out, asinh(t(t(dummy)/sf))/log(2))

    # With subsetting.
    out <- normalizeCounts(dummy, ref, subset_row=1:10)
    sub <- normalizeCounts(dummy[1:10,], ref)
    expect_equal(out, sub)

    out <- normalizeCounts(dummy, subset_row=1:10)
    sub <- normalizeCounts(dummy[1:10,])
    expect_equal(out, sub)

    chosen <- sample(rownames(dummy), 10)
    out <- normalizeCounts(dummy, ref, subset_row=chosen)
    sub <- normalizeCounts(dummy[chosen,], ref)
    expect_equal(out, sub)

    # With subsetting and normalize.all=TRUE.
    out <- normalizeCounts(dummy, subset.row=1:10, normalize.all=TRUE)
    sub <- normalizeCounts(dummy, colSums(dummy[1:10,]))
    expect_equal(out, sub)

    out <- normalizeCounts(dummy, normalize.all=TRUE) # no effect if subset.row=NULL.
    sub <- normalizeCounts(dummy)
    expect_identical(out, sub)

    out <- normalizeCounts(dummy, ref, subset.row=1:10, normalize.all=TRUE) # no effect if sub is supplied.
    sub <- normalizeCounts(dummy, ref)
    expect_identical(out, sub)
})

test_that("method checker does the right thing", {
    library(Matrix)
    setClass("MyDgCMatrix", "dgCMatrix")
    expect_true(scuttle:::.check_methods("none", 1, "MyDgCMatrix", "dgCMatrix"))
    expect_true(scuttle:::.check_methods("log", 1, "MyDgCMatrix", "dgCMatrix"))
    expect_true(scuttle:::.check_methods("log", 10, "MyDgCMatrix", "dgCMatrix"))
    expect_true(scuttle:::.check_methods("asinh", 10, "MyDgCMatrix", "dgCMatrix"))

    setMethod("log1p", "MyDgCMatrix", function(x) {
        cat("YAY!")
        callNextMethod()
    })
    expect_true(scuttle:::.check_methods("none", 1, "MyDgCMatrix", "dgCMatrix"))
    expect_false(scuttle:::.check_methods("log", 1, "MyDgCMatrix", "dgCMatrix"))
    expect_true(scuttle:::.check_methods("log", 10, "MyDgCMatrix", "dgCMatrix"))
    expect_true(scuttle:::.check_methods("asinh", 10, "MyDgCMatrix", "dgCMatrix"))

    setMethod("t", "MyDgCMatrix", function(x) {
        cat("WHEE")
        callNextMethod()
    })
    expect_false(scuttle:::.check_methods("none", 1, "MyDgCMatrix", "dgCMatrix"))
    expect_false(scuttle:::.check_methods("log", 1, "MyDgCMatrix", "dgCMatrix"))
    expect_false(scuttle:::.check_methods("log", 10, "MyDgCMatrix", "dgCMatrix"))
    expect_false(scuttle:::.check_methods("asinh", 10, "MyDgCMatrix", "dgCMatrix"))
})

test_that("normalizeCounts handles silly inputs correctly", {
    out <- normalizeCounts(dummy[0,,drop=FALSE], ref, log=FALSE)
    expect_identical(dim(out), c(0L, as.integer(ncells)))
    out <- normalizeCounts(dummy[0,,drop=FALSE])
    expect_identical(dim(out), c(0L, as.integer(ncells)))
    out <- normalizeCounts(dummy[,0,drop=FALSE], ref[0], log=FALSE)
    expect_identical(dim(out), c(as.integer(ngenes), as.integer(0L)))

    expect_error(normalizeCounts(dummy, ref[0]), "does not equal")
    expect_error(normalizeCounts(dummy, rep(0, ncol(dummy))), "should be positive")
    expect_error(normalizeCounts(dummy, rep(NA_real_, ncol(dummy))), "should be positive")
})

test_that("normalizeCounts behaves with sparse inputs", {
    zeroed <- dummy
    zeroed[rbinom(length(zeroed), 1, 0.95)==1] <- 0

    library(Matrix)
    sparsed <- as(zeroed, "dgCMatrix")
   
    expect_s4_class(out <- normalizeCounts(sparsed, ref), "dgCMatrix")
    expect_equal(normalizeCounts(zeroed, ref), as.matrix(out))

    expect_s4_class(out <- normalizeCounts(sparsed, ref, log=FALSE), "dgCMatrix")
    expect_equal(normalizeCounts(zeroed, ref, log=FALSE), as.matrix(out))

    expect_s4_class(out <- normalizeCounts(sparsed, ref, transform="asinh"), "dgCMatrix")
    expect_equal(normalizeCounts(zeroed, ref, transform="asinh"), as.matrix(out))

    out <- normalizeCounts(sparsed, ref, pseudo.count=2)
    expect_equivalent(as.matrix(normalizeCounts(zeroed, ref, pseudo.count=2)), as.matrix(out))

    expect_s4_class(out <- normalizeCounts(sparsed, ref, subset_row=1:10), "dgCMatrix")
    expect_equal(normalizeCounts(zeroed, ref, subset_row=1:10), as.matrix(out))

    # Trying with a triplet form.
    sparsed <- as(zeroed, "dgTMatrix")
    dimnames(sparsed) <- dimnames(zeroed) # huh, bug in Matrix.
   
    expect_s4_class(out <- normalizeCounts(sparsed, ref), "dgTMatrix")
    expect_equal(normalizeCounts(zeroed, ref), as.matrix(out))

    expect_s4_class(out <- normalizeCounts(sparsed, ref, log=FALSE), "dgTMatrix")
    expect_equal(normalizeCounts(zeroed, ref, log=FALSE), as.matrix(out))

    expect_s4_class(out <- normalizeCounts(sparsed, ref, transform="asinh"), "dgTMatrix")
    expect_equal(normalizeCounts(zeroed, ref, transform="asinh"), as.matrix(out))

    out <- normalizeCounts(sparsed, ref, pseudo.count=2)
    expect_equivalent(normalizeCounts(zeroed, ref, pseudo.count=2), as.matrix(out))

    expect_s4_class(out <- normalizeCounts(sparsed, ref, subset_row=1:10), "dgTMatrix")
    expect_equal(normalizeCounts(zeroed, ref, subset_row=1:10), as.matrix(out))
})

test_that("normalizeCounts behaves with DelayedArray inputs", {
    library(DelayedArray)
    dadum <- DelayedArray(dummy)      
    
    expect_s4_class(out <- normalizeCounts(dadum, ref), "DelayedMatrix")
    expect_equal(normalizeCounts(dummy, ref), as.matrix(out))

    expect_s4_class(out <- normalizeCounts(dadum, ref, log=FALSE), "DelayedMatrix")
    expect_equal(normalizeCounts(dummy, ref, log=FALSE), as.matrix(out))

    expect_s4_class(out <- normalizeCounts(dadum, ref, pseudo.count=2), "DelayedMatrix")
    expect_equal(normalizeCounts(dummy, ref, pseudo.count=2), as.matrix(out))

    expect_s4_class(out <- normalizeCounts(dadum, ref, transform="asinh"), "DelayedMatrix")
    expect_equal(normalizeCounts(dummy, ref, transform="asinh"), as.matrix(out))

    expect_s4_class(out <- normalizeCounts(dadum, ref, subset_row=1:10), "DelayedMatrix")
    expect_equal(normalizeCounts(dummy, ref, subset_row=1:10), as.matrix(out))

    # Library sizes are correctly obtained.
    expect_s4_class(out <- normalizeCounts(dadum), "DelayedMatrix")
    expect_equal(normalizeCounts(dummy), as.matrix(out))

    expect_s4_class(out <- normalizeCounts(dadum, subset_row=1:10), "DelayedMatrix")
    expect_equal(normalizeCounts(dummy, subset_row=1:10), as.matrix(out))

    # Preserves sparsity (or not).
    zeroed <- dummy
    zeroed[rbinom(length(zeroed), 1, 0.95)==1] <- 0
    sparsed <- DelayedArray(as(zeroed, "dgCMatrix"))

    expect_identical(as.matrix(normalizeCounts(sparsed)), as.matrix(normalizeCounts(zeroed)))
    expect_true(is_sparse(normalizeCounts(sparsed)))

    expect_identical(as.matrix(normalizeCounts(sparsed, pseudo.count=2)), as.matrix(normalizeCounts(zeroed, pseudo.count=2)))
    expect_false(is_sparse(normalizeCounts(sparsed, pseudo.count=2)))
})

test_that("normalizeCounts behaves with downsampling", {
    # Testing the two extremes.
    set.seed(1000)
    out <- as.matrix(normalizeCounts(dummy, ref, downsample=TRUE, down.prop=0))
    set.seed(1000)
    tst <- as.matrix(downsampleMatrix(dummy, min(ref)/ref, bycol=TRUE))
    expect_equal(log2(tst+1), out)

    out <- as.matrix(normalizeCounts(dummy, ref, downsample=TRUE, down.prop=1, log=FALSE))
    tst <- as.matrix(normalizeCounts(dummy, ref, log=FALSE))
    expect_identical(out==0, tst==0)
    expect_true(mad(out/tst, na.rm=TRUE) <1e-8)

    # Testing that it actually does the job w.r.t. equalizing coverage.
    lsf <- colSums(dummy)
    out <- normalizeCounts(dummy, lsf, downsample=TRUE, down.prop=0.01, log=FALSE)
    expect_true(mad(colSums(out)) < 1e-8)

    out <- normalizeCounts(dummy, lsf, downsample=TRUE, down.prop=0.05, log=FALSE)
    expect_true(mad(colSums(out)) < 1e-8)

    out <- normalizeCounts(dummy, lsf, downsample=TRUE, down.prop=0.1, log=FALSE)
    expect_true(mad(colSums(out)) < 1e-8)

    # Testing with DelayedArrays. 
    dadum <- DelayedArray(dummy)      
    set.seed(1000)
    out <- normalizeCounts(dummy, ref, downsample=TRUE)
    set.seed(1000)
    tst <- normalizeCounts(dadum, ref, downsample=TRUE)
    expect_equal(out, tst)
})

test_that("normalizeCounts behaves with S(C)E inputs", {
    expect_equivalent(normalizeCounts(counts(X)), 
        normalizeCounts(as(X, "SummarizedExperiment")))

    sf <- runif(ncol(X))
    expect_equivalent(normalizeCounts(counts(X), sf), 
        normalizeCounts(as(X, "SummarizedExperiment"), sf))

    expect_equal(normalizeCounts(counts(X), sizeFactors(X)), normalizeCounts(X))
    expect_equal(normalizeCounts(counts(X), sf), normalizeCounts(X, sf))
})

test_that("logNormCounts works for SE objects", {
    se <- as(X, "SummarizedExperiment")

    cn <- function(se) assay(se, "counts")
    lc <- function(se) assay(se, "logcounts")
    nc <- function(se) assay(se, "normcounts")

    expect_equal(lc(logNormCounts(se)), normalizeCounts(cn(se)))
    expect_equal(nc(logNormCounts(se, log=FALSE)), normalizeCounts(cn(se), log=FALSE))
    expect_equal(lc(logNormCounts(se, pseudo.count=2)), normalizeCounts(cn(se), pseudo.count=2))
    expect_equal(assay(logNormCounts(se, transform="asinh"), "ashcounts"), normalizeCounts(cn(se), transform="asinh"))

    sf <- runif(ncol(se))
    expect_equal(lc(logNormCounts(se, size_factors=sf)),
        normalizeCounts(cn(se), size_factors=sf))
    expect_equal(lc(logNormCounts(se, size_factors=sf, center_size_factors=FALSE)), 
        normalizeCounts(cn(se), size_factors=sf, center_size_factors=FALSE))

    # Subsetting behaves as expected.
    out <- logNormCounts(se, subset.row=1:10)
    ref <- logNormCounts(se[1:10,])
    expect_identical(out, ref)

    out <- logNormCounts(se, subset.row=1:10, normalize.all=TRUE)
    ref <- logNormCounts(se, size.factors=colSums(assay(se)[1:10,]))
    expect_equal(out, ref)

    out <- logNormCounts(se, size.factors=sf, subset.row=1:10)
    expect_identical(out, logNormCounts(se, size.factors=sf)[1:10,])

    # Doesn't break on silly inputs.
    expect_equal(unname(dim(logNormCounts(se[,0,drop=FALSE]))), c(ngenes, 0L))
    expect_equal(unname(dim(logNormCounts(se[0,,drop=FALSE]))), c(0L, ncells)) 
})

test_that("logNormCounts works for SCE objects (basic)", {
    expect_equal(logcounts(logNormCounts(X)), normalizeCounts(counts(X), sizeFactors(X)))
    expect_equal(normcounts(logNormCounts(X, log=FALSE)), normalizeCounts(counts(X), sizeFactors(X), log=FALSE))
    expect_equal(assay(logNormCounts(X, transform="asinh"), "ashcounts"), normalizeCounts(counts(X), sizeFactors(X), transform="asinh"))
    expect_equal(logcounts(logNormCounts(X, pseudo.count=2)), normalizeCounts(counts(X), sizeFactors(X), pseudo.count=2))

    # Checking that size factors are correctly reported.
    Y <- X
    sizeFactors(Y) <- NULL
    Y <- logNormCounts(Y)
    expect_identical(sizeFactors(Y), librarySizeFactors(X))

    sf <- runif(ncol(X))
    Y <- logNormCounts(X, size_factors=sf)
    expect_identical(sizeFactors(Y), sf/mean(sf))

    Y <- logNormCounts(X, size_factors=sf, center_size_factors=FALSE)
    expect_identical(sizeFactors(Y), sf)

    # Checking that my pseudo-count appears and does not overwrite other scater stuff.
    expect_identical(int_metadata(Y)$scater$pseudo.count, 1)

    Z <- X
    int_metadata(Z)$scater <- list(whee="YAY")
    Z <- logNormCounts(Z)
    expect_identical(int_metadata(Z)$scater$pseudo.count, 1)
    expect_identical(int_metadata(Z)$scater$whee, "YAY")

    # Subsetting behaves as expected.
    out <- logNormCounts(X, subset.row=1:10)
    ref <- logNormCounts(X[1:10,])
    expect_identical(out, ref)

    Y <- X
    sizeFactors(Y) <- NULL
    out <- logNormCounts(Y, subset.row=1:10)
    ref <- logNormCounts(Y[1:10,])
    expect_identical(out, ref)

    out <- logNormCounts(Y, subset.row=1:10, normalize.all=TRUE) 
    sub <- logNormCounts(Y, colSums(counts(Y)[1:10,]))
    expect_equal(out, sub)

    # Diverts to other names.
    Y <- logNormCounts(X, name="blah")
    expect_identical(assay(Y, "blah"), logcounts(logNormCounts(X)))
})

test_that("logNormCounts works for SCE objects (altExp)", {
    Y <- X[1:10,]
    counts(Y)[sample(length(counts(Y)))] <- counts(Y) # shuffling for some variety.

    sce <- X
    altExp(sce, "BLAH") <- Y
    sce1 <- applySCE(sce, logNormCounts)

    # Do a class round-trip to wipe out metadata added to the int_* fields.
    COMPFUN <- function(left, right) {
        left <- as(left, "SummarizedExperiment")
        left <- as(left, "SingleCellExperiment")
        sizeFactors(left) <- NULL
        right <- as(right, "SummarizedExperiment")
        right <- as(right, "SingleCellExperiment")
        sizeFactors(right) <- NULL
        expect_equal(left, right)
    }

    COMPFUN(altExp(sce1, "BLAH"), logNormCounts(Y))

    ref <- logNormCounts(sce) # check that it doesn't affect normalization of the main assays.
    altExps(ref) <- NULL
    expect_identical(ref, logNormCounts(X))

    # Other parameters are respected.
    sce3a <- applySCE(sce, logNormCounts, pseudo.count=2)
    COMPFUN(altExp(sce3a), logNormCounts(Y, pseudo.count=2))

    sce3b <- applySCE(sce, logNormCounts, log=FALSE)
    COMPFUN(altExp(sce3b), logNormCounts(Y, log=FALSE))

    # Internal size factors do not propagate to alternative experiments.
    sce4 <- sce
    sizeFactors(sce4) <- runif(ncol(sce))
    sce4 <- applySCE(sce4, logNormCounts)
    COMPFUN(altExp(sce4), logNormCounts(Y))

    # Lack of centering is respected in downstream methods.
    sce5 <- applySCE(sce, logNormCounts, center.size.factors=FALSE)
    COMPFUN(altExp(sce5), logNormCounts(Y, center_size_factors=FALSE))

    # Throws errors with zero-valued size factors.
    sce6 <- sce
    sizeFactors(altExp(sce6)) <- 0
    expect_error(applySCE(sce6, logNormCounts), 'should be positive')
    expect_error(logNormCounts(sce6), NA)
})