Coding potential prediction and classification

coding-potential-prediction-and-classification

Summary

Predicts and classifies RNA sequences as coding or non-coding using computational assessment of protein-coding capacity. This skill is essential for functional annotation of differentially expressed isoforms and long non-coding RNAs in multi-omics pipelines.

When to use

Apply this skill when you have differentially expressed isoform or exon FASTA sequences from transcript assembly or isoform-level analysis (e.g., IsoformSwitchAnalyzer output) and need to distinguish functional protein-coding transcripts from non-coding RNA. Use it as a first step before downstream protein domain or signal peptide annotation.

When NOT to use

• Input sequences are already pre-filtered to known protein-coding genes; redundant classification
• Analyzing short reads or fragments < 200 bp; CPAT reliability decreases on very short sequences
• Non-FASTA input format or sequences without proper isoform/exon identifiers

Inputs

• Differentially expressed isoform FASTA sequences
• Differentially expressed exon FASTA sequences
• IsoformSwitchAnalyzer output FASTA file

Outputs

• CPAT coding potential scores (probability/numeric)
• Binary coding/non-coding classification per isoform
• Unified functional annotation table with isoform identifiers and coding status

How to apply

Load differentially expressed isoform/exon sequences in FASTA format from IsoformSwitchAnalyzer or similar transcript-level output. Run CPAT (Coding Potential Assessment Tool) on these sequences to generate coding potential scores and classify each sequence as coding or non-coding. CPAT uses a machine learning model trained on known coding and non-coding sequences to assign a probability score; sequences are typically classified using a threshold (exact threshold should be specified in pipeline configuration). Merge CPAT coding scores into a unified annotation table indexed by isoform/exon identifier for downstream integration with protein domain (Pfam) and signal peptide (signalP) predictions.

Related tools

• CPAT (Predicts coding potential and classifies sequences as coding or non-coding using machine learning)
• IsoformSwitchAnalyzer (Upstream tool providing differentially expressed isoform/exon FASTA sequences as input to CPAT)
• signalP (Complementary tool for downstream annotation of signal peptides in sequences classified as coding by CPAT)
• Pfam (Complementary tool for protein domain homology detection in CPAT-classified coding sequences)
• Nextflow (Workflow orchestration framework for executing CPAT within multi-omics pipeline) — https://www.nextflow.io

Evaluation signals

• CPAT output file contains valid numeric coding potential scores (0–1 range) for all input isoforms
• Binary classification (coding/non-coding) assigned to every isoform with no missing values
• Unified annotation table row count matches input FASTA sequence count; no sequences dropped
• Isoform identifiers correctly preserved from input FASTA headers through to final annotation table
• CPAT scoring is consistent with downstream Pfam and signalP annotations (e.g., coding isoforms with detected Pfam domains; non-coding sequences lack significant domain hits)

Limitations

• CPAT accuracy depends on machine learning model training; performance may vary for evolutionarily distant or atypical transcript sequences
• Classification threshold is user-configurable but no universal consensus threshold; requires pipeline-specific tuning
• Short sequences (< 200 bp) have reduced CPAT reliability and may yield ambiguous scores near decision boundary
• CPAT does not account for isoform-specific features such as alternative splicing patterns that may affect coding potential assessment

Evidence

• [other] The functional_annotation.nf subworkflow processes differentially expressed isoform sequences by applying CPAT for coding potential assessment: "The functional_annotation.nf subworkflow processes differentially expressed isoform sequences by applying CPAT for coding potential assessment, Pfam for protein domain homology detection, and signalP"
• [other] Load differentially expressed isoform/exon FASTA sequences from IsoformSwitchAnalyzer output and run CPAT to predict coding potential: "Load differentially expressed isoform/exon FASTA sequences from IsoformSwitchAnalyzer output. 2. Run CPAT to predict coding potential and classify sequences as coding or non-coding."
• [other] Merge all annotation results into a unified functional annotation table indexed by isoform/exon identifier: "Merge all annotation results (CPAT coding scores, signalP signal peptide predictions, Pfam domain hits, and motif matches) into a unified functional annotation table indexed by isoform/exon"
• [intro] Pipeline performs functional annotation of transcripts as part of RNAseq analysis: "2. Functional annotation of transcripts"