investigation of (non canonical) splice sites

investigation of (non canonical) splice sites analyzes splice site sequences from genome FASTA files and GFF3 annotations and validates and quantifies canonical and non-canonical splice site occurrences using RNA-Seq read mappings across eukaryotic genomes (plants, fungi, animals).

Key Features:

• Splice Site Analysis: Examines intron removal signals and exon–intron border motifs associated with U2 and U12 spliceosomes to characterize splice site sequences.
• Non-Canonical Splice Sites Identification: Detects and quantifies non-canonical dinucleotide combinations including GC-AG, AT-AC, and other minor variants in addition to canonical GT-AG.
• Cross-Species Conservation and Divergence: Compares splice site patterns across 121 plant genomes and RNA-Seq data from 35 species to assess conservation and true biological divergence from canonical forms.
• Validation via RNA-Seq Data: Uses RNA-Seq read mappings to identify gaps at splice junctions that support the expression of genes containing non-canonical splice sites.
• Comparative Genomic Analysis: Systematically investigates splice site combinations in fungi and animals, highlighting patterns such as increased CT-AC frequency in fungal genomes and GA-AG occurrences in Eurytemora affinis and Oikopleura dioica.
• Splice Site Flexibility: Measures relative flexibility of splice site positions and reports a generally higher variability at the 3' splice site compared to the 5' splice site across kingdoms.

Scientific Applications:

• Genomic Annotation and Correction: Identifies non-canonical splice sites and potential annotation errors to support refinement of genome annotations.
• Evolutionary Biology Studies: Provides comparative data on splice site conservation and divergence to inform studies of evolutionary pressures on splicing mechanisms.
• Functional Genomics: Validates non-canonical splice site usage with RNA-Seq to support analyses of alternative splicing and its contribution to proteomic diversity.

Methodology:

Performs genome sequence analysis of FASTA files and GFF3 annotations, annotation review, RNA-Seq read mapping to validate splice junctions and quantify canonical and non-canonical splice site usage, and generates comprehensive reports and visualizations.