title: Transforming microbial genotyping: a robotic pipeline for genotyping bacterial strains.
abstract: Microbial genotyping increasingly deals with large numbers of samples, and data are commonly evaluated by unstructured approaches, such as spread-sheets. The efficiency, reliability and throughput of genotyping would benefit from the automation of manual manipulations within the context of sophisticated data storage. We developed a medium- throughput genotyping pipeline for MultiLocus Sequence Typing (MLST) of bacterial pathogens. This pipeline was implemented through a combination of four automated liquid handling systems, a Laboratory Information Management System (LIMS) consisting of a variety of dedicated commercial operating systems and programs, including a Sample Management System, plus numerous Python scripts. All tubes and microwell racks were bar-coded and their locations and status were recorded in the LIMS. We also created a hierarchical set of items that could be used to represent bacterial species, their products and experiments. The LIMS allowed reliable, semi-automated, traceable bacterial genotyping from initial single colony isolation and sub-cultivation through DNA extraction and normalization to PCRs, sequencing and MLST sequence trace evaluation. We also describe robotic sequencing to facilitate cherrypicking of sequence dropouts. This pipeline is user-friendly, with a throughput of 96 strains within 10 working days at a total cost of < €25 per strain. Since developing this pipeline, >200,000 items were processed by two to three people. Our sophisticated automated pipeline can be implemented by a small microbiology group without extensive external support, and provides a general framework for semi-automated bacterial genotyping of large numbers of samples at low cost.
authors.affiliation: Environmental Research Institute, University College Cork, Cork, Ireland. b.ofarrell@ucc.ie
authors.forename: Brian
authors.initials: B
authors.lastname: O'Farrell
authors.forename: Jana K
authors.initials: JK
authors.lastname: Haase
authors.forename: Vimalkumar
authors.initials: V
authors.lastname: Velayudhan
authors.forename: Ronan A
authors.initials: RA
authors.lastname: Murphy
authors.forename: Mark
authors.initials: M
authors.lastname: Achtman
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics*; D015373:Bacterial Typing Techniques / Q000295:instrumentation / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005838:Genotype; D060005:Genotyping Techniques / Q000295:instrumentation / Q000379:methods*; D058885:Multilocus Sequence Typing / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D012984:Software
chemical_list: D004269:DNA, Bacterial