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Transforming microbial genotyping : a robotic pipeline for genotyping bacterial strains
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Watson, Michael, O’Farrell, Brian, Haase, Jana K., Velayudhan, Vimalkumar, Murphy, Ronan A. and Achtman, Mark (2012) Transforming microbial genotyping : a robotic pipeline for genotyping bacterial strains. PLoS ONE, 7 (10). e48022. doi:10.1371/journal.pone.0048022
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Official URL: http://dx.doi.org/10.1371/journal.pone.0048022
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.
| Item Type: | Journal Article | ||||
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| Divisions: | Faculty of Medicine > Warwick Medical School > Biomedical Sciences > Microbiology & Infection Faculty of Medicine > Warwick Medical School > Biomedical Sciences > Translational & Experimental Medicine Faculty of Medicine > Warwick Medical School |
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| Journal or Publication Title: | PLoS ONE | ||||
| Publisher: | Public Library of Science | ||||
| ISSN: | 1932-6203 | ||||
| Official Date: | 29 October 2012 | ||||
| Dates: |
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| Volume: | 7 | ||||
| Number: | 10 | ||||
| Page Range: | e48022 | ||||
| DOI: | 10.1371/journal.pone.0048022 | ||||
| Status: | Peer Reviewed | ||||
| Publication Status: | Published | ||||
| Access rights to Published version: | Open Access | ||||
| Funder: | Science Foundation Ireland (SFI) | ||||
| Grant number: | 05/FE1/B882 |
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