High Throughput Screening In The Manchester Laboratory

1. High Throughput Screening In The Manchester Laboratory Chris Charlton, St. Mary’s Hospital

2. Introduction • Worked for Regional Genetics Service at St. Mary’s for 3 Years • Primary Role is High-Throughput Screening of BRCA1/2, MLH1, MSH2 & CFTR • Pre-Analytical Duties

3. Optimised Procedures • Primer Dispense • PCR Setup • PCR Product and Sequencing Purification • Sequence Data Organisation

4. BRCA1 CF BRCA2 MLH1 MSH2 Our Section • Rotation Between Diseases • Important that System is Flexible • Focus of Talk is Working Example of a BRCA2 Screen

5. BRCA2 • Samples Referred through Clinical Genetics and sent from Liverpool • 15 Patients per Batch • 8 Week Turnaround

6. Exons - 2, 17, 4, 7, 5+6, 8 Exons – 9, 19, 12, 13, 14, 18 Exons – 15, 26, 16, 20, 25, 21 Exons - 11a - f Fragment 4 - (Exon 22/23/24), Exons 10a, 10b, 27, 3 5 x 96 Well Plate Setup

7. Primer Dispense Deck Setup Tip Box Primer Source Plate • The Robot dispenses 2µl of primer into each well from a source plate • Primers oven dried at 50°C and stored at -20°C Destination Plates

8. PCR Setup Tip Boxes Source Plates • Uses DNA source, dried down primer and custom PCR mastermix plates • Robot dispenses 18µl of mastermix and 2µl of DNA per well • PCR conditions - 60°C (exon 11), 55°C (rest) Primer Plates Other Tip Boxes delivered via Cytomat conveyor belt

9. PCR/Sequencing Purification • Purified using Agencourt Ampure and Cleanseq protocols • Carried out using post PCR 96 Tip head Robots

10. Also robot can dispense sequence mastermix (bigdye/N13) into 10 plates (forward and reverse sequence) Plates are diluted using MilliQ water according to gel document Plates run on ABI 3730 Result of procedures amounts to 870 ABI sequence traces to be organised and analysed

11. Data Analysis – QC Check • Identifies signal strength of data and other anomalous trends Good Strong Very Poor Weak

12. Sequencing Repeats • QC stage essential for quick turnaround of repeat data • List of potential sequence or PCR repeats compiled into excel spreadsheet

13. Breakdown of Repeat Rates • Re-Electrophoresis 40-80 • Re-Sequence 15-30 • Re-PCR 5-10 • Out of 870 sequence files represents a 5-10% repeat rate for whole screen

14. Sequence Arrangement • ABI files from 3730 ordered by patient lab number using in-house software • Files can be arranged by any chosen identifier along with a control

16. MLH1 MLH1 MLH1 CFTR CFTR CFTR BRCA2 BRCA2 BRCA2 Data Storage CMMC Genetics Server Data Repository • Our genetics server R: is the repository where data is stored • Separate folders for progress, gene, batch and patient Completed Data New Data Data In Progress ABI Raw Data Batch/Patient Folders

17. Genotyping Data • Using Pregap and Gap4 software to analyse whole patient data • Any positives are added to repeat list as confirmation PCR

18. Mutation Surveyor • Sequence Traces also ran through Mutation Surveyor, and tablature output is checked to ensure nothing has been missed

19. Data Distribution • Whole patients are distributed to a number of clinical scientists for validation • Quick turnaround for bulks of data via home-working policy

20. Audit Trail • Contains information about batch makeup, process dates, positive results and validation information. • Colour coding indicates reporting status

21. Conclusion • Automation and Data analysis methods described ensure a high level of efficiency and accuracy of a patient screen at all stages. • Principles can be applied to other diseases

22. Acknowledgements • Jo Brock • Megan Adaway • Claudia Largan • Emma Howard • Andrew Wallace • Stuart Bayliss • Rob Elles