ACACIA

ACACIA performs high-accuracy allele calling from Illumina amplicon sequencing data to genotype complex multigene systems and mitigate PCR and sequencing artifacts.

Key Features:

• High Allele Calling Accuracy: Achieved allele calling accuracy exceeding 98% in validation experiments.
• Artifact Management: Distinguishes chimeric and non-chimeric PCR/sequencing artifacts and characterizes their behavior relative to copy number variation (non-chimeric artifacts increase linearly with CNV; chimeric artifacts stabilize above 4–6 co‑amplified alleles).
• Amplification Efficiency Analysis: Accounts for heterogeneous amplification efficiencies among allelic variants when co‑amplifying multiple loci.
• Benchmarking: Compared against previously published pipelines and demonstrated enhanced reliability and precision in allele calling.

Scientific Applications:

• Genotyping complex multigene systems: Applied to genotype novel complex multigene systems, particularly in non‑model organisms where co‑amplification artifacts complicate inference.
• Validation on Gallus gallus: Validated using chicken (Gallus gallus) with 43 artificial genotypes containing 2–13 alleles per amplicon.
• Simulation of non-model conditions: Evaluated using "naive" primer design based on closely related Galliform species to simulate primer mismatches and non‑model species scenarios.
• CNV and artifact interpretation: Provides empirical insight into how artifact types relate to copy number variation to inform interpretation in high‑CNV datasets.

Methodology:

Processes Illumina amplicon sequencing data through a pipeline that calls alleles while minimizing PCR and sequencing artifacts, compares performance to previously published pipelines, and investigates the relationship between artifacts and copy number variation (CNV).