Non-canonical ORFs GENCODE Phase II ncORFs Prim Track Settings
Assembly: Human Dec. 2013 (GRCh38/hg38) Data last updated at UCSC: 2026-05-19 11:39:16
Description
The three Gencode ncORF tracks in the non-canonical ORF track container show
non-canonical translated open reading frames (ncORFs) identified
from ribosome profiling (Ribo-seq) data and mapped to the GENCODE annotation by the
GENCODE / TransCODE consortium.
The data is available in two phases:
Phase I
The Phase I catalog contains 7,264 unique human ncORFs called from Ribo-seq data
across seven publications and mapped to GENCODE v35. Only translations of 16 codons or above
and initiating from ATG start codons were incorporated. Redundant sense-overlapping ORFs were
merged. Of these, 3,085 ORFs were found by more than one publication, providing independent
replication evidence. This catalog was developed as part of an effort to standardize the
annotation of translated ORFs across reference databases including Ensembl/GENCODE, HGNC,
UniProtKB, and PeptideAtlas.
Phase II
The Phase II catalog nearly quadruples the Phase I set, defining 28,359 ncORFs in the
Comprehensive set, mapped to GENCODE v45. Compared to Phase I, additional published
Ribo-seq datasets were incorporated and the restrictions on ORF size and initiation codon
were lifted.
Two subsets are provided for the Phase II data:
Comprehensive (28,359 ncORFs) – all mapped translations from the expanded catalog
Primary (10,127 ncORFs) – a high-confidence subset filtered for translations
with especially robust translation signatures, as extrapolated from Ribo-seq data.
These ncORFs demonstrate translation evidence comparable to canonical protein-coding genes.
Display Conventions and Configuration
All three GENCODE ncORF tracks are displayed in bigGenePred format and labeled with their
ORF identifier. The default color scheme and available filter controls differ by track.
Phase I — Kozak strength colors (default)
The Phase I track colors items by Kozak consensus strength by default.
Two alternative color schemes can be selected from the track controls page
(Color by dropdown): Evidence type and HLA class (see below).
Golden amber — Strong Kozak context. Both position −3 (A/G) and
position +4 (G) match the consensus.
Steel blue — Moderate Kozak context. One of the two positions matches.
Gray — Weak Kozak context. Neither position matches.
Black — Non-ATG start codon (Kozak rule does not apply) or context
unavailable.
Phase I — Evidence type colors (alternative)
Select Color by: Evidence type to highlight peptide evidence from
Deutsch et al. (see References). ORFs with no mass spectrometry evidence are gray.
Gold — TransCODE peptidein (628 ORFs). Confirmed as
confidently translated by PeptideAtlas; candidate for peptidein annotation in
reference databases.
Steel blue — HLA immunopeptidomics evidence only (1,373 ORFs).
Forest green — Non-HLA (whole-cell tryptic) evidence only (66 ORFs).
Orange — Both HLA and non-HLA evidence (35 ORFs).
Gray — No peptide evidence (5,114 ORFs) or in the peptidein set
based on binding predictions only with no direct MS sequences (48 ORFs).
Phase I — HLA class colors (alternative)
Select Color by: HLA class to color items by the HLA class in which peptides were
detected:
Steel blue — Class I only (1,632 ORFs).
Crimson — Class II only (10 ORFs).
Orange — Both class I and class II (143 ORFs).
Gray — No HLA data (5,479 ORFs).
Phase I — Filters
The Phase I track can be filtered by: start codon, Kozak strength, Kozak TE, replicated
status, and — using the peptide evidence fields — peptidein status
(isPeptidein), HLA class (hlaClass), HLA evidence tier
(hlaFinalTier), HPP guideline category (hlaHppCategory), and Ribo-seq
quality (riboseqQuality).
Mouseover for Phase I shows ORF name, host gene, Kozak strength and TE,
replicated status, peptidein flag, HLA evidence tier, and HLA peptide count.
Phase II — colors and filters
The Phase II Primary and Comprehensive tracks color items by Kozak strength using the
same scheme as Phase I. Peptide evidence fields are not included in the Phase II tracks.
Common filters: start codon, Kozak strength, Kozak TE.
Peptide evidence fields (Phase I only)
Each Phase I item carries the following peptide evidence fields from Deutsch et al.
(2026), accessible via the details page and Table Browser:
Field
Description
isPeptidein
yes/no: ORF is in the PeptideAtlas peptidein set (Table S12)
The raw data can be explored interactively with the
Table Browser or the
Data Integrator. The data can be accessed from
scripts through our API; the track names are
"gencNcOrfs" (Phase I), "gencNcOrfsPrimary" (Phase II Primary),
and "gencNcOrfsComprehensive" (Phase II Comprehensive).
For automated download and analysis, the genome annotations are stored in bigBed files that
can be downloaded from
our download server.
Individual regions or the whole genome annotation can be obtained using our tool
bigBedToBed, which can be compiled from the source code or downloaded as a precompiled
binary for your system. Instructions for downloading source code and binaries can be found
here.
Methods
Phase I Ribo-seq catalog
Mudge et al. (2022, see References) consolidated translation evidence from seven published
ribosome profiling datasets that used harringtonine or lactimidomycin treatment to enrich
for translation initiation sites. Ribo-seq reads were mapped to the GENCODE v35 annotation
on GRCh38. Only ATG-initiated ORFs of at least 16 codons were retained, and redundant
sense-overlapping ORFs were merged by taking the longest representative, yielding 7,264
ncORFs across five biotype classes: upstream ORFs (uORFs), downstream ORFs (dORFs),
intronic ORFs (intORFs), pseudogenic translations (PT), and lncRNA-embedded ORFs.
The catalog was developed as part of a reference-database coordination effort involving
Ensembl/GENCODE, HGNC, UniProtKB, and PeptideAtlas.
Phase II Ribo-seq catalog
Chothani et al. (2025, see References) expanded the catalog by incorporating additional
Ribo-seq datasets across more cell types and tissues and mapping to GENCODE v45. The
ATG-start codon and 16-codon length restrictions were lifted to capture near-cognate
initiations and micropeptides. A data-driven scoring framework using ribosome occupancy
uniformity and P-site in-frame fraction identified a Primary subset of 10,127 ncORFs with
translation signatures comparable to canonical coding genes; the Comprehensive set contains
all 28,359 mapped ORFs.
Kozak strength and translational efficiency
Each ORF was annotated with its Kozak consensus strength by fetching the 11-base genomic
context around the start codon from hg38.2bit and classifying positions −3 and +4
relative to the A of the start codon: both matching (A/G at −3 and G at +4) =
Strong; one matching = Moderate; neither = Weak; non-ATG start = non-ATG. A numeric
translational efficiency (TE) score was also assigned by looking up the 11-base context in
the Noderer 2014 TE table (Mol Syst Biol 10:748, PMID 25170020).
Peptide evidence and peptideins
Deutsch et al. (2026, see References) queried the 7,264 Phase I ORFs against two independent
PeptideAtlas mass spectrometry repositories. The HLA immunopeptidomics build was constructed
from HLA-I and HLA-II peptidomes across more than 100 HLA-typed donors spanning multiple
tissue types and cancer cell lines; peptides were enriched by affinity purification and
identified by tandem mass spectrometry. The whole-cell tryptic proteomics build used
conventional shotgun proteomics from a broad range of cell lines and tissues. Spectra
were manually reviewed and classified according to the Prensner et al. tier system (Tier 1B =
numerous HPP-quality HLA peptides; Tier 2B = a single qualifying HLA peptide; Tier 1A/2A =
additional non-HLA evidence). The study introduced the term peptidein for a
translation product detectable by mass spectrometry but not yet annotatable as a protein due
to absent functional evidence. Of the 7,264 Phase I ORFs, 628 passed PeptideAtlas curation
as peptideins (Table S12); a further 1,522 have HLA or tryptic peptide evidence below the
peptidein threshold.
The supplementary data tables (Tables S2, S3, S6, S7, and S12) from Deutsch et al. were
downloaded from the paper's supplementary materials at
https://doi.org/10.1038/s41586-026-10459-x.
Each table was joined to the Phase I bigGenePred by the short ORF identifier (e.g.,
c14riboseqorf80) using the script
addPeptideEvidence.py.
The script appended 14 new fields to all 7,264 Phase I items; the 5,114 ORFs without peptide
evidence receive default empty values so they remain visible in the track and filterable on
isPeptidein and related fields. Non-HLA peptides that map to known proteins or
are too short to be informative were excluded (Tables S2, exclude column).
The complete build procedure is documented in
ncOrfs.txt.
Credits
Thanks to Jonathan Mudge, Jorge Ruiz-Orera, John Prensner, Sebastiaan van Heesch, and the
GENCODE / TransCODE consortium for creating and maintaining these annotations.