reassignTSSbyCage: Reassign all Transcript Start Sites (TSS)

In Roleren/ORFik: Open Reading Frames in Genomics

Reassign all Transcript Start Sites (TSS)

Description

Given a GRangesList of 5' UTRs or transcripts, reassign the start sites using max peaks from CageSeq data. A max peak is defined as new TSS if it is within boundary of 5' leader range, specified by 'extension' in bp. A max peak must also be higher than minimum CageSeq peak cutoff specified in 'filterValue'. The new TSS will then be the positioned where the cage read (with highest read count in the interval). If removeUnused is TRUE, leaders without cage hits, will be removed, if FALSE the original TSS will be used.

Usage

reassignTSSbyCage(
  fiveUTRs,
  cage,
  extension = 1000,
  filterValue = 1,
  restrictUpstreamToTx = FALSE,
  removeUnused = FALSE,
  preCleanup = TRUE,
  cageMcol = FALSE
)

Arguments

fiveUTRs	(GRangesList) The 5' leaders or full transcript sequences
cage	Either a filePath for the CageSeq file as .bed .bam or .wig, with possible compressions (".gzip", ".gz", ".bgz"), or already loaded CageSeq peak data as GRanges or GAlignment. NOTE: If it is a .bam file, it will add a score column by running: convertToOneBasedRanges(cage, method = "5prime", addScoreColumn = TRUE) The score column is then number of replicates of read, if score column is something else, like read length, set the score column to NULL first.
extension	The maximum number of basses upstream of the TSS to search for CageSeq peak.
filterValue	The minimum number of reads on cage position, for it to be counted as possible new tss. (represented in score column in CageSeq data) If you already filtered, set it to 0.
restrictUpstreamToTx	a logical (FALSE). If TRUE: restrict leaders to not extend closer than 5 bases from closest upstream leader, set this to TRUE.
removeUnused	logical (FALSE), if False: (standard is to set them to original annotation), If TRUE: remove leaders that did not have any cage support.
preCleanup	logical (TRUE), if TRUE, remove all reads in region (-5:-1, 1:5) of all original tss in leaders. This is to keep original TSS if it is only +/- 5 bases from the original.
cageMcol	a logical (FALSE), if TRUE, add a meta column to the returned object with the raw CAGE counts in support for new TSS.

Details

Note: If you used CAGEr, you will get reads of a probability region, with always score of 1. Remember then to set filterValue to 0. And you should use the 5' end of the read as input, use: ORFik:::convertToOneBasedRanges(cage) NOTE on filtervalue: To get high quality TSS, set filtervalue to median count of reads overlapping per leader. This will make you discard a lot of new TSS positions though. I usually use 10 as a good standard.

TIP: do summary(countOverlaps(fiveUTRs, cage)) so you can find a good cutoff value for noise.

Value

a GRangesList of newly assigned TSS for fiveUTRs, using CageSeq data.

See Also

Other CAGE: assignTSSByCage(), reassignTxDbByCage()

Examples

# example 5' leader, notice exon_rank column
fiveUTRs <- GenomicRanges::GRangesList(
  GenomicRanges::GRanges(seqnames = "chr1",
                         ranges = IRanges::IRanges(1000, 2000),
                         strand = "+",
                         exon_rank = 1))
names(fiveUTRs) <- "tx1"

# make fake CAGE data from promoter of 5' leaders, notice score column
cage <- GenomicRanges::GRanges(
  seqnames = "1",
  ranges =  IRanges::IRanges(500, width = 1),
  strand = "+",
  score = 10) # <- Number of tags (reads) per position
# notice also that seqnames use different naming, this is fixed by ORFik
# finally reassign TSS for fiveUTRs
reassignTSSbyCage(fiveUTRs, cage)
# See vignette for example using gtf file and real CAGE data.

Roleren/ORFik documentation built on Nov. 13, 2024, 10 p.m.