filterHits: Filters framecalled data based on the mean number of hits...
In riboSeqR: Analysis of sequencing data from ribosome profiling experiments

Description Usage Arguments Details Value Author(s) See Also Examples

If no ribosomal footprints of the correct lengths (and frames) are seen at a coding sequence in any replicate group, this sequence is unlikely to be translated (and is therefore likely to be uninteresting). This function filters out these coding sequences, leaving only those with a minimum number of hits in at least one replicate group, and a minimum number of unique sequences aligning in at least one replicate group (to exclude single stacks of sequenced reads passing the filtering).

1 2	filterHits(fCs, lengths = 27, frames, hitMean = 10, unqhitMean = 1, ratioCheck = TRUE, fS)

`fCs`	`riboCoding` object defining the number of ribosome footprint reads observed over a set of coordinates, their lengths, and their frame relative to coding start sites.
`lengths`	The lengths of ribosome footprint reads to be used in filtering.
`frames`	If given, the frames of the ribosome footprint reads to be used in filtering. Should be of equal length to the ‘lengths’ parameter - see examples.
`hitMean`	The mean number of hits within a replicate group that should be observed to pass filtering.
`unqhitMean`	The mean number of unique sequences within a replicate group that should be observed to pass filtering. This parameter is intended to avoid cases where a coding sequence is deemed to be expressed based on a few highly expressed sequences.
`ratioCheck`	Checks the ratios of expected phase to maximal phase within the putative coding sequence. See Details.
`fS`	The output of 'frameCounting' function. Required if 'ratioCheck = TRUE'.

Frames can be given as a single vector (which specifies the frames used for all lengths of footprints, or as a list, specifying the frame for each length given in ‘lengths’.

For highly translated coding regions, the small proportion of out-of-phase reads may cause overlapping but out-of-phase putative coding regions to pass this filtering step. If 'ratioCheck = TRUE', filterHits identifies those cases where the phase with the maximum number of reads (the maximal phase) is not the expected phase for that putative coding region. If the ratio of reads in the expected phase to maximal phase does not significantly (Chi-squared test with significance threshold of 0.05) exceed that ratio observed for all coding regions, the putative coding region is filtered out.

riboCoding object containing the filtered coding sequences and the associated numbers of ribosome footprint reads.

Thomas J. Hardcastle

frameCounting

#ribosomal footprint data
datadir <- system.file("extdata", package = "riboSeqR")
ribofiles <- paste(datadir, 
                   "/chlamy236_plus_deNovo_plusOnly_Index", c(17,3,5,7), sep = "")
rnafiles <- paste(datadir, 
                  "/chlamy236_plus_deNovo_plusOnly_Index", c(10,12,14,16), sep = "")

riboDat <- readRibodata(ribofiles, rnafiles, replicates = c("WT", "WT",
"M", "M"))

# CDS coordinates
chlamyFasta <- paste(datadir, "/rsem_chlamy236_deNovo.transcripts.fa", sep = "")
fastaCDS <- findCDS(fastaFile = chlamyFasta, 
                    startCodon = c("ATG"), 
                    stopCodon = c("TAG", "TAA", "TGA"))

# frame calling
fCs <- frameCounting(riboDat, fastaCDS)

# analysis of frame shift for 27 and 28-mers.
fS <- readingFrame(rC = fCs, lengths = 27:28)

# filter coding sequences. 27-mers are principally in the 1-frame,
# 28-mers are principally in the 0-frame relative to coding start (see
# readingFrame function).

ffCs <- filterHits(fCs, lengths = c(27, 28), frames = list(1, 0), 
                   hitMean = 50, unqhitMean = 10, fS = fS)