Automated workflow for genotyping individual transposon library variants.

Background: The fields of genome engineering and synthetic biology rely on high-throughput screening to accelerate research and increase reproducibility. This often requires the use of costly equipment such as flow cytometers, specialized microfluidic devices or liquid handlers. Despite the availability of population-level screening methods, genotyping individual clones in transposon libraries remains a necessary but frequently labor-intensive task well suited for liquid-handling platforms. Although open-source automation is democratizing access to liquid handlers, it requires a minimum level of programming expertise or the availability of validated protocols to implement experimental workflows. To address these challenges, we present a step-by-step automated workflow designed for individual variant genotyping of transposon insertion libraries using an open-source OT-2 liquid handler and a custom Python package to annotate sequencing results. Methods: The automated protocol comprises six modular steps that can be used separately or integrated into different workflows: 1) colony picking into selective media, 2) counter-selection using the backbone and integrated antibiotic resistances and preparation of glycerol stocks pre-cultures, 3) colony counter-selection and 4) control PCR assays to filter spurious integration events, 5) arbitrary PCR assays and 6) sequencing and annotation using a custom Python package. This protocol is demonstrated using standardized vectors adhering to the Standard European Vector Architecture (SEVA) guidelines and standardized Lab Automation Protocol (LAP) scripts to ensure reproducibility and portability. Results: The automated workflow was validated using a transposon insertion library in Pseudomonas putida KT2440, enabling reliable genotyping of individual variants within 4-5 days. No spurious integrations were detected, and genomic insertion sites were successfully identified for 40 out of 46 variants, demonstrating robustness, accessibility, and scalability of the open-source workflow. Discussion: This automated workflow enables reliable genotyping while reducing hands-on time and minimizing human error. Its modular, standardized design and validated protocols promote protocol reuse and sharing while broadening access to open-source laboratory automation. While the throughput of the workflow is limited by the deck capacity of the liquid handler, it can be scaled by subsequent runs or parallel processing on multiple liquid handlers. In addition, the workflow can be further streamlined through integration of an OT-2 adapted colony picker. Overall, this open-source workflow provides a robust foundation for scalable and reproducible genotyping of individual variants in genome engineering and synthetic biology applications.