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