Amino Acid Variation

Methodology for Determining Amino Acid Variation

1. Feature Selection
   AAV-Atlas focuses on genomic features that encode amino acids. These features are identified based on metadata tags (CODES_AMINO_ACIDS) in the AAV-Atlas database. Each feature is associated with:

   • A reference genome (REF_MASTER_AAV2), which serves as the standard for comparison.
   • A set of aligned sequences from various sources, including public repositories like GenBank.

2. Alignment and Sequence Processing
   The analysis begins by processing multiple sequence alignments:

   • Alignments are organized hierarchically. Only tip alignments---those without child alignments---are considered, ensuring the analysis targets the finest resolution of aligned sequences.
   • For each alignment, the script processes member sequences corresponding to the feature of interest.

3. Codon and Amino Acid Comparison
   For every sequence within the alignment:

   • Amino acid residues are retrieved for each codon in the selected feature.
   • The retrieved residues are compared against those encoded in the reference genome.

4. Replacement Identification
   A replacement is recorded when:

   • The amino acid in the sequence differs from the reference residue.
   • The codon does not contain ambiguous nucleotides (N), or the replacement is unambiguously a single amino acid residue.
   • Both the reference and replacement residues are biologically meaningful (e.g., not * or X).

5. Annotation of Replacements
   Each replacement is annotated with:

   • The feature it belongs to.
   • The codon position and corresponding nucleotide sequence.
   • The reference amino acid and replacement amino acid.
   • A unique identifier (feature:refAA:codonLabel:replacementAA) for traceability.

6. Biochemical Classification
   Replacements are further classified based on their biochemical properties using established metrics:

   • Hanada's Radical/Conservative Categories (2006): Assesses changes in biochemical properties such as charge and polarity.
   • Grantham Distance (1974): Quantifies the biochemical distance between two amino acids.
   • Miyata Distance (1979): Measures the evolutionary distance between amino acids.

7. Database Integration
   Identified replacements are stored in custom tables within AAV-Atlas:

   • The primary table (aav_replacement) catalogs the replacements and their associated metadata.
   • A secondary table (aav_replacement_sequence) links replacements to the sequences in which they were observed.

8. Variation Creation
   Each replacement is linked to a variation object in the database, enabling downstream analyses such as:

   • Visualization: Mapping replacements to protein structures or genome browsers.
   • Queries: Retrieving replacements by feature, codon, or biochemical classification.
   • Phylogenetic Analysis: Studying replacement patterns across evolutionary lineages.

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