Exploring Amino Acid Variation

Overview

AAV-Atlas provides a comprehensive framework for exploring amino acid (AA) variation across adeno-associated virus (AAV) sequences. This section outlines how to examine AA replacements and their frequencies within aligned datasets.

AAV-Atlas leverages the GLUE bioinformatics framework to analyze genetic variation at the amino acid level. Since all sequences in AAV-Atlas are typed by species and serotype, the data is hierarchically organized into tip alignments corresponding to specific AAV types (e.g., AAV-10). Each tip alignment is constrained by a reference sequence, allowing AA variations to be mapped directly to a defined context.

Identifying Amino Acid Replacements

To begin exploring AA-level genetic variation, the following command lists observed AA replacements along with their counts:

list custom-table-row aav_replacement id reference_aa codon_label replacement_aa num_seqs

Command Breakdown:

• custom-table-row: This GLUE command lists rows from a custom table (in this case, 'aav_replacement').

• id: A unique identifier for each replacement event.

• reference_aa: The amino acid found in the reference sequence at a given codon position.

• codon_label: The codon position (in the alignment) where the replacement is observed.

• replacement_aa: The observed replacement amino acid.

• num_seqs: The number of sequences exhibiting this replacement.

Example Output:

Mode path: /project/aav
GLUE> list custom-table-row aav_replacement id reference_aa codon_label replacement_aa num_seqs
+=================================+==============+=============+================+==========+
|               id                | reference_aa | codon_label | replacement_aa | num_seqs |
+=================================+==============+=============+================+==========+
| REF_AAV10:PLA2:A:24:E           | A            | 24          | E              | 1        |
| REF_AAV10:PLA2:P:5:L            | P            | 5           | L              | 35       |
| REF_AAV10:Rep40:D:205:G         | D            | 205         | G              | 17       |
| REF_AAV10:Rep40:F:348:L         | F            | 348         | L              | 17       |
| REF_AAV10:Rep40:R:409:W         | R            | 409         | W              | 1        |
| REF_AAV10:Rep40:T:257:A         | T            | 257         | A              | 34       |
| REF_AAV10:Rep52:D:205:G         | D            | 205         | G              | 17       |
| REF_AAV10:Rep52:F:348:L         | F            | 348         | L              | 17       |
| REF_AAV10:Rep52:T:257:A         | T            | 257         | A              | 34       |
| REF_AAV10:Rep68:D:429:G         | D            | 429         | G              | 17       |
| REF_AAV10:Rep68:T:481:A         | T            | 481         | A              | 34       |
| REF_AAV10:Rep78:D:429:G         | D            | 429         | G              | 17       |
| REF_AAV10:Rep78:F:572:L         | F            | 572         | L              | 17       |
| REF_AAV10:Rep78:T:481:A         | T            | 481         | A              | 34       |
| REF_AAV10:VP1:A:589:T           | A            | 589         | T              | 196      |
| REF_AAV10:VP1:A:67:E            | A            | 67          | E              | 1        |
| REF_AAV10:VP1:D:24:A            | D            | 24          | A              | 1        |

In this example:

• At codon 205 in the Rep68 protein of AAV-10, 17 sequences show a replacement of the reference amino acid (D) with glycine (G).

• At codon 589 in the VP1 (capsid) protein of AAV-10, 196 sequences show alanine (A) replaced by threonine (T).

Calculating Amino Acid Frequencies

To assess the relative frequency of AA replacements within a specific alignment, use the following command:

Mode path: /project/aav
GLUE> alignment AL_AAV10 amino-acid frequency -r REF_AAV10 -f X -l 122 122

Command Breakdown:

• alignment AL_AAV10: Specifies the alignment to analyze (in this case, the AAV-10 alignment).

• amino-acid frequency: Calculates the frequency of amino acids at a specific codon position.

• -r REF_AAV10: Indicates the reference sequence for AAV-10.

• -f X: Specifies the feature (e.g., a protein or region) to focus on.

• -l 122 122: Limits the analysis to codon position 122.

Example Output:

| feature | codon | aminoAcid | numMembers | pctMembers |
|---------|-------|-----------|------------|------------|
| X       | 122   | K         | 19         | 8.84       |
| X       | 122   | R         | 196        | 91.16      |

In this example:

• At codon 122, lysine (K) is found in 8.84% of sequences, while arginine (R) is observed in 91.16% of sequences.

Interpreting the Results

The combination of AA replacement listings and frequency calculations provides powerful insights into genetic variability across AAV sequences. This information can be valuable for:

• Understanding Diversity: By examining frequent AA replacements, researchers can identify regions under selective pressure or regions of functional significance.

• Vector Engineering: Insights into conserved and variable regions of AAV capsids can inform the design of chimeric vectors or therapeutic vectors.

• Tracking Evolution: Observing shifts in AA frequencies over time or across serotypes can shed light on the evolutionary trajectories of AAV lineages.

Alignment Hierarchy and Context

AAV-Atlas organizes sequences hierarchically based on serotype and species. Sequences typed as AAV-10, for example, are assigned to the AAV-10 tip alignment, constrained by the AAV-10 reference genome. This hierarchical approach ensures that observed AA variations are context-specific, enhancing the precision and relevance of the analysis.

By leveraging these commands, researchers can systematically investigate amino acid variation, unlocking valuable insights into AAV biology and its applications in gene therapy and virology research.

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