a-o-tool

a-o-tool refines protein sequences by integrating orthology information with de novo transcriptome assembly to improve accuracy and completeness of target sequences for drug discovery.

Key Features:

• Orthology-Based Sequence Validation: Compares human Swiss-Prot protein sequences with sequences from other species to validate and refine targets using orthologous relationships.
• De Novo Transcriptome Assembly: Performs de novo assembly of transcriptomes from paired-end RNA-Seq to generate full-coverage cDNA sequences for tissues across human, mouse, rat, dog, pig, and cynomolgus monkey.
• Automated Workflow: Uses reciprocal BLAST searches to identify best-matching orthologous hits, extracts sequences, translates them to proteins, and performs multiple sequence alignments.
• Quantitative Refinement Metrics: Computes metrics quantifying sequence identity and coverage to measure the degree of sequence refinement achieved.
• High Hit Rates and Coverage: Reports empirical hit rates greater than 60% for human assemblies with instances of 100% sequence coverage and identity and higher sequence identities observed in cynomolgus monkey.
• Public Database Comparison: Addresses incomplete or erroneous entries in public databases such as Ensembl, UniProt, and RefSeq by comparing assembled and refined sequences against these sources.

Scientific Applications:

• Drug-target sequence refinement: Improves the accuracy of protein sequences used to study drug–target interactions in vitro.
• Recombinant protein generation: Provides full-coverage cDNA-derived protein sequences to support production of high-fidelity recombinant proteins.
• Cross-species orthology assessment: Reduces misinterpretation from gaps or errors in non-human orthologous sequences when extrapolating experimental results across species.

Methodology:

De novo transcriptome assembly from paired-end RNA-Seq; reciprocal BLAST searches comparing assembled sequences to human Swiss-Prot and public databases (Ensembl, UniProt, RefSeq); extraction and translation of best hits to protein sequences; multiple sequence alignment; and computation of sequence identity and coverage metrics.