Molecular Ecology Resources

Abstract

Genotyping by amplicon sequencing (GBAS) is a relatively low-cost approach for generating genotypic data compared with established genomic methods, making it highly scalable and particularly suitable for large-scale genetic monitoring projects.However, most existing analytical pipelines are either marker-specific, insufficiently scalable, or lacking efficient data management systems for the long-term integration of genotypic information, limiting the full potential of GBAS.Here, we address this gap by introducing GBAS-GUI (https:// github. com/ sonne nbe-dot/ GBAS-GUI), a pipeline capable of generating GBAS-based genotypic data for a wide variety of loci at scale.GBAS-GUI integrates a graphical user interface with multiple checkpoints to improve accessibility and robustness.It implements multiprocessing architecture and a relational database that links genotypic data with associated sample metadata to enhance scalability and data management.The pipeline further enables marker screening through automated calculation of polymorphism information content (PIC) and implements a strategy to recover homologous genotypic information from paralogous loci with non-overlapping amplicon length ranges.Using multiple empirical datasets, we demonstrate substantial improvements in processing speed, database management and handling artefacts related to co-amplification of unspecific regions and duplicates of the same genomic region.We further show that incorporating the full sequence information captured by an amplicon increases marker information content beyond what is achievable with length-based genotyping alone and expands the analytical versatility of GBAS.Overall, GBAS-GUI provides a robust, scalable and versatile framework that unlocks the potential of GBAS for large-scale population genetic and phylogeographic studies. | IntroductionGenotyping-by-sequencing (GBS) involves the use of highthroughput sequencing (HTS) technologies to produce largescale genotypic data.Although GBS is better known through methods like RAD-sequencing, which allow researchers to obtain genome-wide genotypic information for non-model species (Baird et al. 2008), it also includes amplicon sequencing (Tibihika et al. 2020).This approach involves sequencing Polymerase Chain Reaction (PCR) products targeting specific