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
DNA Extractor
Barcoding
Liquid handling robots
Sequencer
LIMS
TaqMan
Process redesign
Pre White Paper Post White Paper
Kit car Toyota production line
Toyota Production System: Principles of lean thinking
Specify value
Create flow
Reduce waste
(muda)
Monitor
performance
PCR set up
Pre White paper Post White paper
Batching approach Production line4 or 8 patients per gene Any number of patients, any gene
PCR set up
(a) Large genes/medium throughput
eg. BRCA1/2
(b) “Normal traffic”Need to use common PCR mix and thermocycler program
PCR workflow
298 amplicons7 thermocycler programs
5 PCR mixes
MegaMix Royal
60°C anneal426/432 amplicons
Kit car
Toyota
How many water blanks?
One water blank per amplicon
?
Kit car
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%)
96-well sequencing pipeline
Manual PCR
by scientists and technologists
Sequencing
by one technologist
ExoSAP/DyeEx3730
UnidirectionalExon ± 10bp
Mutation Surveyor + visual inspection
DNA extraction process
DNA Extraction
DNA Quantification
Samples in 2D bar coded tubes
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
Robotic PCR workflow
DNA in 2D barcoded tubes Check rack
layoutNormalised DNA plate (96 well)
Primer dilutions in 2D barcoded tubes
Check rack layout PCR plates (4x96 well)
MegaMix
384-well sequencing pipeline
Robotic PCR
Band 4 technologists
Sequencing
Band 4 technologist
Agencourt3730
UnidirectionalExon ~-50 to +10bp
Mutation Surveyor (semi-automated)
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
Mutation report
HGVS (Quality) report
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
Aim of increased laboratory automation
Safer systems
Increased efficiency
Increased capacity
Activity data 2001- 2008
0
2000
4000
6000
8000
10000
12000
14000
01-02 02-03 03-04 04-05 05-06 06-07 07-08
Num
ber Samples
Reports
Genotypes
92% of genotypes are now generated by sequencing (vs 36% in 2001-2002)
0
500
1000
1500
2000
2500
2005-2006 2006-2007 2007-2008 2008-2009Projected
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
40 days (no. of reports)
40 days (% within target)
Reporting time data 2005 - 2009
Implementation of laboratory automation
DNA Extractor
Barcoding
Liquid handling robots
Sequencer
TaqMan
SCOBEC Genetics Laboratories Network
Molecular genetic testing
Past Present Future