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Open Reading Frames of Transcripts from Their Sequences

Source: R/orfs.R
transcript_orfs.Rd

Computes, for every assayed transcript, an open reading frame (ORF) and the sequence-level features that decide whether it is productive. Requires transcript sequences in the annotation directory (see extract_transcript_sequences()). The ORF of each transcript comes from the first applicable source (orf_source):

  1. "annotation": a complete annotated CDS. Its stop codon is added when the annotation excludes it (as GENCODE does) and the ORF is re-read from the annotated start codon to the first in-frame stop, so a CDS that does not match the sequence is corrected (annotation_cds_agrees = FALSE). Transcripts tagged seleno, ribosomal_slippage or non_ATG_start keep the annotated CDS.

  2. "projected": the start codon of the gene's canonical isoform lies in an exon of the transcript and reads ATG there; translation proceeds from that position to the first in-frame stop (the ORFanage approach). This is what rescues novel isoforms without CDS.

  3. "annotation_partial": an annotated CDS flagged incomplete (cds_start_NF / cds_end_NF) whose start could not be projected; the annotated span is kept, and when its 3' end is known the NMD rule still applies (GENCODE annotates many NMD transcripts this way).

  4. "longest": none of the above; the longest ATG-to-stop ORF of at least min_orf_length nt is used and coding_probability tells whether it is believable.

  5. "none": no ORF of at least min_orf_length nt.

From the ORF the function derives the stop codon (NA when translation runs into the transcript end, a non-stop decay substrate), the UTR lengths, uORFs in the 5' UTR (n_uorf, complete upstream ORFs; n_overlapping_uorf, out-of-frame upstream ORFs overlapping the main start), the genomic coordinates of start and stop, whether start and stop are shared with the canonical isoform and whether the reading frame at the stop codon is in frame with the canonical ORF (frame_vs_canonical), and the NMD class:

  • "ptc": the stop lies more than nmd_rule nt upstream of the last exon-exon junction (the 50-nt rule), NMD predicted.

  • "ptc_start_proximal": same, but the ORF is shorter than start_proximal nt; still called unproductive, with the note that such transcripts can escape NMD by translation re-initiation.

  • "non_stop": no in-frame stop codon.

  • "long_3utr": no premature stop, but a 3' UTR longer than long_utr3 nt (default Inf, so the flag is off unless asked for).

  • "none": no NMD feature.

  • NA: no stop codon in a transcript whose 3' end is annotated as incomplete (cds_end_NF / mRNA_end_NF, column end_incomplete), so nothing can be said.

coding_potential() scores every transcript's longest ORF with the Fickett TESTCODE statistic and an in-frame hexamer usage score, combined by a logistic model trained on GENCODE coding versus lncRNA transcripts (see data-raw/build_coding_model.R); coding_probability above coding_cutoff (0.364, the CPAT human cutoff) is called coding.

Usage

transcript_orfs(
  self,
  gene_name = NULL,
  min_orf_length = 90L,
  nmd_rule = 50,
  start_proximal = 150,
  long_utr3 = Inf,
  ...
)

coding_potential(
  self,
  gene_name = NULL,
  min_orf_length = 90L,
  coding_cutoff = 0.364,
  ...
)

Arguments

self

An IsoformicExperiment or an annotation directory path.

gene_name

Optional genes (names or IDs) to restrict the result to.

min_orf_length

Minimum ORF length in nt, stop codon included (default 90, i.e. 29 amino acids).

nmd_rule

Distance (nt) from the stop codon to the last junction above which NMD is predicted; default 50.

start_proximal

ORF length (nt) below which a premature stop is classed ptc_start_proximal instead of ptc; default 150.

long_utr3

3' UTR length (nt) above which long_3utr is flagged; default Inf.

...

Passed on to methods.

coding_cutoff

Coding probability above which a transcript is called coding (default 0.364).

Value

A tibble with one row per transcript.

See also

transcript_productivity(), transcript_features()

Examples

iso <- example_isoformic()
orfs <- transcript_orfs(iso, gene_name = "RBPJ")
#> Warning: ! 1 transcript has a sequence length differing from the exon length; its ORF is
#>   skipped.
orfs[, c("transcript_name", "orf_source", "orf_length", "nmd_class", "coding_probability")]
#> # A tibble: 10 × 5
#>    transcript_name orf_source         orf_length nmd_class coding_probability
#>    <chr>           <chr>                   <dbl> <chr>                  <dbl>
#>  1 RBPJ-222        annotation_partial        283 NA                    0.0723
#>  2 RBPJ-201        annotation               1503 none                  0.975 
#>  3 RBPJ-206        annotation               1503 none                  0.984 
#>  4 RBPJ-225        projected                1140 none                  0.950 
#>  5 RBPJ-204        annotation               1464 none                  0.980 
#>  6 RBPJ-205        annotation               1461 none                  0.968 
#>  7 RBPJ-214        annotation               1398 none                  0.976 
#>  8 RBPJ-208        annotation               1149 none                  0.931 
#>  9 RBPJ-202        annotation               1461 none                  0.968 
#> 10 RBPJ-210        longest                   462 none                  0.548