Firth Software Tools

Firth Software Tools estimate completeness and diversity of protein-encoding libraries for directed evolution experiments using statistical analyses of mutagenesis-generated variant collections.

Key Features:

• Library completeness and diversity estimation: Estimates completeness and diversity of protein-encoding libraries generated by oligonucleotide-directed mutagenesis, error-prone polymerase chain reaction (epPCR), DNA shuffling, StEP PCR, synthetic shuffling, and other random mutagenesis methods.
• Statistical algorithms: Uses equations derived from simple statistics to estimate expected completeness and to predict required library sizes to achieve specified probabilities of full coverage.
• Program modules: Provides PEDEL (estimates distinct variants in epPCR libraries), DRIVeR (diversity statistics for shuffled libraries), GLUE-Including Translation (GLUE-IT) (predicts expected amino acid completeness for libraries with up to six independently varied codons and user-specified randomization schemes), CodonCalculator (maps randomized codons to amino acids), and AA-Calculator (identifies degenerate codons encoding specified amino acid sets).
• Amino acid-level analysis (PEDEL-AA): Calculates amino acid statistics for epPCR-derived libraries using parent sequence, mutation rate, library size, indel rates, and nucleotide mutation matrices to report protein-level completeness and diversity.
• Premature termination codon analysis: Reports counts and length distributions of sequences truncated by premature termination codons.

Scientific Applications:

• Directed evolution library design: Informs mutagenesis strategy and library sizing to maximize capture of protein variant space for directed evolution experiments.
• Protein engineering assessment: Evaluates DNA- and protein-level diversity to support evolution of properties such as increased stability or novel enzymatic activities.

Methodology:

Performs statistical analysis using equations derived from simple statistics and nucleotide mutation matrices to compute expected completeness and diversity; accounts for parent sequence, mutation rate, library size, and indel rates; computes expected amino acid completeness for up to six independently varied codons and reports variant counts and length distributions of sequences truncated by premature termination codons; implemented in C++ source code.