In order to find a threshold value to filter lowly expressed features, SeqGate analyzes the distribution of counts found in replicates along with zero counts. More specifically, features with a customizable minimal proportion of zeros in one condition are selected. The distribution of counts found in replicates of that same condition along with those zeros is computed. The chosen threshold is the count value corresponding to the customizable percentile of this distribution. Finally, features having a customizable proportion (90% by default) of replicates with counts below that value in all conditions are filtered. Default value for all customizable parameters have been set through extensive simulation batch testing and can be considered as adequate in most situations.
To install SeqGate, start R and enter:
if (!requireNamespace("BiocManager", quietly = TRUE)) install.packages("BiocManager") BiocManager::install("SeqGate")
First load SeqGate:
The main input data is a [SummarizedExperiment] (https://bioconductor.org/packages/release/bioc/html/ SummarizedExperiment.html) object which contains an assay with count data. Briefly, a SummarizedExperiment container contains one or more assays, each represented by a matrix-like object of numeric or other mode. The rows typically represent genomic ranges of interest and the columns represent samples. For SeqGate, the SummarizedExperiment object must contain at least one assay of numeric counts, and a DataFrame describing the columns, in particular a column telling the biological condition the sample belongs to. To apply SeqGate, the SummarizedExperiment object, the assay name and the column describing the condition of each sample in the colData dataframe, must be given.
Let's load some toy data set. This data set is an extract from a human transcriptome dataset produced by Strub et al. (2011), in which human cells expressing the Microphtalmia Transcription Factor (MiTF) are compared to cells in which the MiTF is repressed. The extract counts 1,000 genes with expression measured in 3 samples for each biological condition (the full table of read counts is available in the Supplementary materials of Dillies, M.A. et al. (2012)).
And now we define a vector indicating the biological condition corresponding to each column of data_MiTF_1000. Here the two biological conditions are 'A' and 'B'.
The toy dataset that we have just loaded is not yet a SummarizedExperiment object, such as required in Seqgate input. We thus need to create it, from the count matrix and the biological condition annotation.
rowData <- DataFrame(row.names=rownames(data_MiTF_1000genes)) colData <- DataFrame(Conditions=cond) counts_strub <- SummarizedExperiment( assays=list(counts=data_MiTF_1000genes), rowData=rowData, colData=colData)
By default, SeqGate only needs the SummarizedExperiment object along with the name of the assay we want to work with, and the name of the column which contains the biological conditions annotation. Thus, we can apply the SeqGate method filtering, by calling the following code:
counts_strub <- applySeqGate(counts_strub,"counts","Conditions")
As a result, the input SummarizedExperiment object now includes a new column in the rowData DataFrame, named onFilter. This column is a logical vector that indicates if the gene should be kept after filtering (TRUE) or not (FALSE). The metadata of the object also include a new element, named "threshold", which gives the value of the applied threshold.
Thus, to get the matrix of features intended to be kept for the downstream analysis:
keptGenes <- assay(counts_strub[rowData(counts_strub)$onFilter == TRUE,]) head(keptGenes) dim(keptGenes)
To get the applied threshold:
We can also get the matrix of filtered genes:
filteredOut <- assay(counts_strub[rowData(counts_strub)$onFilter == FALSE,]) head(filteredOut)
To conclude, we can see that, from the initial set of 1,000 genes,
r nrow(keptGenes) have been kept, after the application of a threshold of
r metadata(counts_strub)$threshold: all genes having less than
replicates with less than
r metadata(counts_strub)$threshold reads are
Besides the three mandatory parameters described above, the applySeqGate function also have three other parameters, that can be set to refine the filtering:
By default, 'prop0' is set to the maximum number of replicates minus one, divided by the maximum number of replicates. In the example above, as we have 3 replicates in both conditions, the maximum number of replicates is 3. Thus, the parameter 'prop0' is set to 2/3. This means that we consider that the gene is lowly expressed if it has 2 zeros among its 3 replicates.
The distribution of maximum counts from all the lowly expressed genes (selected
according to 'prop0') is then computed. The idea is to see how high a count can
be in a replicate alongside a zero in another replicate. In order to introduce
flexibility, we do not simply take the maximum count of the distribution but a
'percentile' of this distribution. By default, when the number of replicates in
at least one condition is below 5, 'percentile' is set to 0.9. In the above
example, the 90th percentile of the distribution of maximum counts seen
alongside a zero is
r metadata(counts_strub)$threshold, and this is the
threshold that we will apply in order to actually filter the lowly expressed
Finally, the filter is applied according to a last parameter: propUpThresh.
SeqGate does keep those genes whose counts are above the computed threshold in
at least 'propUpThresh' replicates, in at least one condition. Still in the
example used precedently, this means that all genes whose counts are above
r metadata(counts_strub)$threshold in 3 x 0.9 = 2.7 replicates, are kept.
As it is not possible to consider 2.7 replicates, the value is rounded to the
next integer, that is 3 in this case. Finally in this example, a gene is kept if
all its 3 replicates have a count above
in at least one condition.
Default value for all customizable parameters have been set through extensive simulation batch testing and can be considered as adequate in most situations. However, one may consider that the default parameters are not suited to its experiment. In that case, custom values can be given:
counts_strub <- applySeqGate(counts_strub,"counts","Conditions", prop0=1/3, percentile=0.8, propUpThresh=0.5)
This time, from the initial set of 1,000 genes,
r nrow(assay(counts_strub[rowData(counts_strub)$onFilter == TRUE,])) have been kept,
after the application of a threshold of
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