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    R script to filter probesets with log-expression values below the lowest spike-in

    27th January 2010

    Sometimes there is a need to remove all the probesets, which have expression values below the minimal spike-in intensity on the Affymetrix microarray. The reasoning behind this procedure is simple: minimal-expression spike-ins represent the bottom margin of microarray sensitivity, and anything below that margin cannot be reliably quantified – which also means that both fold-change and p-value of expression variance will be unreliable for these probesets.

    Here’s a simple R script to do just that. It is abundantly commented, and also contains an optional (commented out) fragment which allows the removal of more low-variance, low-intensity probesets.


    Hint: click the “plain text” box header to be able to right-click the code, “Select All”, and then “Copy”.
    [CODE]
    filter_below_spikes = function(eset) {
    # Finds max(lowest) AFFX/spike-in intensity, and removes rows consisting entirely of values below max(lowest).
    # @param eset
    # ExpressionSet
    # @returns
    # exprs(ExpressionSet), filtered

    # Without Biobase exprs() will not work.
    require(Biobase)
    expr = exprs(eset)

    # ‘expr’ sample:
    # 1
    # 1367452_at 10.880208
    # 1367453_at 10.554647

    cat(nrow(expr), “rows before filtering.\n”)

    # Make a vector of spike row names.
    spikes = grep(“AFFX”, rownames(expr), value = TRUE)
    cat(“Expression matrix has”, length(spikes), “spike-in rows.\n”)
    cat(“Summary of spike-in values distribution follows:\n”)
    print(summary(expr[spikes, ]))

    # Find max(lowest) spike-in values.
    minval_max = max(as.double(substr(grep(“Min”, summary(expr[spikes, ]), value = TRUE), 10, 14)))
    cat(“max(minimal spike-in log-intensity values) =”, minval_max, “\n”)

    # Remove spike-ins from expr.
    expr = expr[grep("AFFX", rownames(expr), value = TRUE, invert = TRUE), ]

    nospike_rows = nrow(expr)
    cat(nospike_rows, “rows remaining after the removal of”, length(spikes), “spike-in probesets.\n”)

    # Optional: calculate max(SD) of all removed rows.
    #bad_sds_max = max(apply(expr[!apply((expr > minval_max), 1, any),], 1, sd))

    # Now remove all rows, where each value is <= minval_max. expr = expr[!apply((expr <= minval_max), 1, all), ] cat(nrow(expr), "rows remaining after filtering out", nospike_rows - nrow(expr), "probesets with all values below", minval_max, "\n") #cat(bad_sds_max, "is max(SD) of all", nospike_rows - nrow(expr), "filtered probesets with all values below", minval_max, "\n") # Optional: Remove *some* of the rows, which have at least one value below minval_max, and row_SD <= bad_sds_max. #pre_final_rows = nrow(expr) #expr = expr[(apply(expr, 1, sd) > bad_sds_max) | (apply(expr, 1, min) > minval_max), ]
    #cat(pre_final_rows-nrow(expr), “rows with SD <=", bad_sds_max, "and min(row) <=", minval_max, "were removed.\n") #cat(nrow(expr), "final rows returned.\n") return(expr) } [/CODE] Sample use:

    > source(“script.R”)
    > expr.filtered = filter_below_spikes(eset)

    Comments and suggestions are welcome.

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