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Laboratory Automation

From kit car to Toyota production line; process redesign in the Exeter laboratory Sian Ellard, Carolyn Tysoe, Martina Owens, Melissa Sloman, Kevin Colclough, Andrew Parrish, Neil Goodman, Karen Stals, Michael Day, Katie Guegan, Ann-Marie Patch and Beverley Shields. Laboratory Automation.

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Laboratory Automation

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  1. From kit car to Toyota production line; process redesign in the Exeter laboratorySian Ellard, Carolyn Tysoe, Martina Owens, Melissa Sloman, Kevin Colclough, Andrew Parrish, Neil Goodman, Karen Stals, Michael Day, Katie Guegan, Ann-Marie Patch and Beverley Shields

  2. Laboratory Automation Barcoding DNA Extractor Liquid handling robots TaqMan LIMS Sequencer

  3. Process redesign Pre White Paper Post White Paper Kit car Toyota production line

  4. Toyota Production System: Principles of lean thinking • Specify value • Create flow • Reduce waste (muda) • Monitor performance

  5. PCR set up Pre White paper Post White paper Batching approach Production line 4 or 8 patients per gene Any number of patients, any gene

  6. PCR set up (a) Large genes/medium throughput eg. BRCA1/2 (b) “Normal traffic” Need to use common PCR mix and thermocycler program

  7. PCR workflow 7 thermocycler programs 298 amplicons Kit car 5 PCR mixes MegaMix Royal 60°C anneal Toyota 426/432 amplicons

  8. How many water blanks? Kit car One water blank per amplicon ? Toyota • Water blank contamination for manual PCRs assessed by gel electrophoresis 1/685 (0.15%) • Water blank contamination rate for PCRs (manual and robotic) assessed by gel electrophoresis and sequencing; 0/1647 (<0.07%)

  9. 96-well sequencing pipeline Sequencing by one technologist Manual PCR by scientists and technologists ExoSAP/DyeEx 3730 Unidirectional Exon ± 10bp Mutation Surveyor + visual inspection

  10. DNA extraction process • Gentra installed March 2007 • DNA stored in 2D barcoded tubes since March 2007 • Software for barcode checking of samples on/off Gentra October 2008; Excel installed January 2009 and system implemented in February DNA Extraction DNA Quantification Samples in 2D bar coded tubes

  11. Robotic PCR workflow DNA in 2D barcoded tubes Check rack layout Normalised DNA plate (96 well) Primer dilutions in 2D barcoded tubes Check rack layout PCR plates (4x96 well) MegaMix

  12. 384-well sequencing pipeline Sequencing Band 4 technologist Robotic PCR Band 4 technologists Agencourt 3730 Unidirectional Exon ~-50 to +10bp Mutation Surveyor (semi-automated)

  13. Semi-automated sequence analysis • Sensitivity of unidirectional analysis for heterozygous substitutions is >99.6% (n=701 heterozygous base substitutions) CMGS study; Genetic Testing and Molecular Biomarkers In press • Inspect mutation report to confirm mutations and polymorphisms (delete false positive calls) • Inspect HGVS table to check ROIs covered, ROI quality scores meet threshold and inspect bases with a PHRED-like score <20

  14. Mutation report

  15. HGVS (Quality) report

  16. Rationalisation of workflows • Triplet repeats – rationalised within SCOBEC • LightCycler assays (FVL, prothrombin and HFE) to TaqMan. Robotic setup and result generated from software. • Agarose gel assays (B27 and JAK2) replaced by fluorescent PCR (added to HNF1A c.872dup assay). Robotic setup and result generated from GeneMarker software. • Manual tests: Haemato-oncology, CFTR OLA and MLPA

  17. Aim of increased laboratory automation • Safer systems • Increased efficiency • Increased capacity

  18. Activity data 2001- 2008 92% of genotypes are now generated by sequencing (vs 36% in 2001-2002)

  19. Reporting time data 2005 - 2009

  20. Implementation of laboratory automation Barcoding DNA Extractor Liquid handling robots TaqMan Sequencer

  21. SCOBEC Genetics Laboratories Network Past Present Future Molecular genetic testing

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