The genFeatures function generates a variety of feature types from TxDb objects using utilities provided by the GenomicFeatures package.

Obtain feature types

The first step is the generation of the feature type ranges based on annotations provided by a GFF file that can be transformed into a TxDb object. This includes ranges for mRNAs, exons, introns, UTRs, CDSs, miRNAs, rRNAs, tRNAs, promoter and intergenic regions. In addition, any number of custom annotations can be included in this routine.

txdb <- makeTxDbFromGFF(file = "data/tair10.gff", format = "gff3", 
    organism = "Arabidopsis")
feat <- genFeatures(txdb, featuretype = "all", reduce_ranges = TRUE, 
    upstream = 1000, downstream = 0, verbose = TRUE)

Count and plot reads of any length

The featuretypeCounts function counts how many reads in short read alignment files (BAM format) overlap with entire annotation categories. This utility is useful for analyzing the distribution of the read mappings across feature types, e.g. coding versus non-coding genes. By default the read counts are reported for the sense and antisense strand of each feature type separately. To minimize memory consumption, the BAM files are processed in a stream using utilities from the Rsamtools and GenomicAlignment packages. The counts can be reported for each read length separately or as a single value for reads of any length. Subsequently, the counting results can be plotted with the associated plotfeaturetypeCounts function.

The following generates and plots feature counts for any read length.

outpaths <- subsetWF(args, slot = "output", subset = 1, index = 1)
fc <- featuretypeCounts(bfl = BamFileList(outpaths, yieldSize = 50000), 
    grl = feat, singleEnd = TRUE, readlength = NULL, type = "data.frame")
p <- plotfeaturetypeCounts(x = fc, graphicsfile = "results/featureCounts.png", 
    graphicsformat = "png", scales = "fixed", anyreadlength = TRUE, 
    scale_length_val = NULL)

Figure 2: Read distribution plot across annotation features for any read length.

Count and plot reads of specific lengths

To determine the approximate read length of ribosome footprints in Ribo-Seq experiments, one can generate and plot the feature counts for specific read lengths separately. Typically, the most abundant read length obtained for translated features corresponds to the approximate footprint length occupied by the ribosomes.

fc2 <- featuretypeCounts(bfl = BamFileList(outpaths, yieldSize = 50000), 
    grl = feat, singleEnd = TRUE, readlength = c(74:76, 99:102), 
    type = "data.frame")
p2 <- plotfeaturetypeCounts(x = fc2, graphicsfile = "results/featureCounts2.png", 
    graphicsformat = "png", scales = "fixed", anyreadlength = FALSE, 
    scale_length_val = NULL)

Figure 3: Read distribution plot across annotation features for specific read lengths.

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