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Poly(A) Sites and Alternative Polyadenylation of Transcripts

Source: R/polyadenylation.R
polyadenylation.Rd

polya_sites() describes the 3' end of every assayed transcript:

  • tes_pos, tes_group (alternative 3' ends of the gene, from transcript_features()), tes_rank (1 = most proximal 3' end along the direction of transcription), n_tes_groups, is_proximal, is_distal.

  • apa_type relative to the canonical isoform: "same_end", "tandem_utr" (different 3' end, same stop codon: 3' UTR shortening or lengthening), "alternative_last_exon" (different last exon), "intronic" (3' end inside an intron of the canonical isoform, typically a composite terminal exon) or "other".

  • utr3_length (from transcript_orfs() when sequences are available, else from the annotated CDS).

  • polya_signal and polya_signal_distance: the poly(A) signal hexamer (AATAAA, ATTAAA or one of the weaker variants) found in the last signal_window nt of the transcript sequence, and its distance to the 3' end (canonical signals sit 10-30 nt upstream); NA without sequences.

  • With polya_atlas, the nearest atlas site within window bp of the 3' end: pas_site_id, pas_class, pas_tpm, pas_distance, pas_supported. The atlas is the frozen PolyASite 2.0 table (polya_atlas = "polyasite", see frozen_data), a data frame with seqid, cleavage_pos, strand (and optionally site_id, site_class, tpm), or a GENCODE polyAs.gff3.gz file (download_reference(file_type = "polya")).

apa_usage() quantifies per gene, for genes with at least two 3' end groups: the share of expression ending at the distal-most 3' end (distal_fraction) and the abundance-weighted 3' UTR length (weighted_utr3_length), per sample or per group with the change between the last and the first group level. test_apa() tests the change in distal usage between two groups with the quasi-binomial test used by test_productive_fraction(), on counts.

Usage

polya_sites(
  self,
  gene_name = NULL,
  polya_atlas = NULL,
  window = 50L,
  signal_window = 50L,
  ...
)

read_polya_gff(path)

apa_usage(
  self,
  group = NULL,
  levels = NULL,
  assay = "abundance",
  min_expr = 0,
  as_matrix = FALSE,
  gene_name = NULL,
  ...
)

test_apa(
  self,
  group,
  levels = NULL,
  assay = "counts",
  min_expr = 0,
  gene_name = NULL,
  ...
)

Arguments

self

An IsoformicExperiment.

gene_name

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

polya_atlas

NULL (no atlas), "polyasite", a data frame or a file path (Parquet table or GENCODE poly(A) GFF3).

window

Distance in bp within which an atlas site supports a 3' end (default 50).

signal_window

Length of the 3' terminal sequence searched for a poly(A) signal (default 50 nt).

...

Passed on to methods.

path

Path to a GENCODE polyAs.gff3.gz file.

group

Name of a col_data column defining groups; NULL for per-sample values.

levels

Order of the group levels; the change is last - first. For the tests (test_productive_fraction(), test_apa()), the two groups to compare; samples of other groups are ignored.

assay

Transcript assay to use ("abundance" for usage, "counts" for the test).

min_expr

Minimum assay value for an isoform to count (default 0).

as_matrix

Logical, return the genes x samples matrix of distal fractions instead of the summary table.

Value

Tibbles with one row per transcript (polya_sites()) or per gene (apa_usage(), test_apa()); a matrix for as_matrix = TRUE.

Examples

iso <- example_isoformic()
pa <- polya_sites(iso, gene_name = "RBPJ")
#> Warning: ! 1 transcript has a sequence length differing from the exon length; its ORF is
#>   skipped.
pa[, c("transcript_name", "tes_rank", "apa_type", "utr3_length", "polya_signal")]
#> # A tibble: 10 × 5
#>    transcript_name tes_rank apa_type              utr3_length polya_signal
#>    <chr>              <int> <chr>                       <dbl> <chr>       
#>  1 RBPJ-222               1 alternative_last_exon          NA NA          
#>  2 RBPJ-201               6 same_end                     4121 NA          
#>  3 RBPJ-206               3 tandem_utr                      0 NA          
#>  4 RBPJ-225               2 alternative_last_exon         347 NA          
#>  5 RBPJ-204               4 tandem_utr                     59 AAAAAG      
#>  6 RBPJ-205               6 same_end                     4121 NA          
#>  7 RBPJ-214               5 tandem_utr                    176 NA          
#>  8 RBPJ-208               5 other                         357 NA          
#>  9 RBPJ-202               6 same_end                     4124 NA          
#> 10 RBPJ-210               5 tandem_utr                     NA NA          
head(apa_usage(iso, group = "condition"))
#> # A tibble: 6 × 13
#>   gene_id        gene_name n_tes_groups apa_types mean_gene_expr distal_fraction
#>   <chr>          <chr>            <int> <chr>              <dbl>           <dbl>
#> 1 ENSG000000094… REV3L                2 intronic           831.            0.647
#> 2 ENSG000001308… PNCK                 4 alternat…           50.6           0.594
#> 3 ENSG000001001… POLR2F               6 alternat…          350.            0.125
#> 4 ENSG000001005… PSMA3                6 alternat…          860.            0.686
#> 5 ENSG000000703… FGF22                2 intronic            48.2           0.364
#> 6 ENSG000000542… ARID4B               7 alternat…         1116.            0.703
#> # ℹ 7 more variables: weighted_utr3_length <dbl>,
#> #   distal_fraction_preeclampsia <dbl>, distal_fraction_control <dbl>,
#> #   delta_distal_fraction <dbl>, weighted_utr3_length_preeclampsia <dbl>,
#> #   weighted_utr3_length_control <dbl>, delta_weighted_utr3_length <dbl>
head(test_apa(iso, group = "condition"))
#> # A tibble: 6 × 12
#>   gene_id            gene_name distal_fraction_preeclam…¹ distal_fraction_cont…²
#>   <chr>              <chr>                          <dbl>                  <dbl>
#> 1 ENSG00000009413.16 REV3L                         0.752                  0.593 
#> 2 ENSG00000048828.17 FAM120A                       0.920                  0.961 
#> 3 ENSG00000100038.19 TOP3B                         0.925                  0.976 
#> 4 ENSG00000074319.13 TSG101                        0.0104                 0.0208
#> 5 ENSG00000146648.18 EGFR                          0.924                  0.990 
#> 6 ENSG00000100142.15 POLR2F                        0.223                  0.0834
#> # ℹ abbreviated names: ¹​distal_fraction_preeclampsia, ²​distal_fraction_control
#> # ℹ 8 more variables: delta_distal_fraction <dbl>, log_odds_ratio <dbl>,
#> #   se <dbl>, statistic <dbl>, dispersion <dbl>, df <dbl>, pvalue <dbl>,
#> #   padj <dbl>