PMID- 23144721
OWN - NLM
STAT- MEDLINE
DCOM- 20140826
LR  - 20211021
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 7
IP  - 10
DP  - 2012
TI  - Transforming microbial genotyping: a robotic pipeline for genotyping bacterial 
      strains.
PG  - e48022
LID - 10.1371/journal.pone.0048022 [doi]
LID - e48022
AB  - 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 < euro25 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.
FAU - O'Farrell, Brian
AU  - O'Farrell B
AD  - Environmental Research Institute, University College Cork, Cork, Ireland. 
      b.ofarrell@ucc.ie
FAU - Haase, Jana K
AU  - Haase JK
FAU - Velayudhan, Vimalkumar
AU  - Velayudhan V
FAU - Murphy, Ronan A
AU  - Murphy RA
FAU - Achtman, Mark
AU  - Achtman M
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20121029
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 0 (DNA, Bacterial)
SB  - IM
MH  - Bacteria/classification/*genetics
MH  - Bacterial Typing Techniques/instrumentation/*methods
MH  - DNA, Bacterial/chemistry/genetics
MH  - Genotype
MH  - Genotyping Techniques/instrumentation/*methods
MH  - Multilocus Sequence Typing/instrumentation/*methods
MH  - Polymerase Chain Reaction
MH  - Reproducibility of Results
MH  - Sequence Analysis, DNA
MH  - Software
PMC - PMC3483277
COIS- Competing Interests: The authors have declared that no competing interests exist.
EDAT- 2012/11/13 06:00
MHDA- 2014/08/27 06:00
PMCR- 2012/10/29
CRDT- 2012/11/13 06:00
PHST- 2012/07/11 00:00 [received]
PHST- 2012/09/20 00:00 [accepted]
PHST- 2012/11/13 06:00 [entrez]
PHST- 2012/11/13 06:00 [pubmed]
PHST- 2014/08/27 06:00 [medline]
PHST- 2012/10/29 00:00 [pmc-release]
AID - PONE-D-12-20112 [pii]
AID - 10.1371/journal.pone.0048022 [doi]
PST - ppublish
SO  - PLoS One. 2012;7(10):e48022. doi: 10.1371/journal.pone.0048022. Epub 2012 Oct 29.