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Streamlining of PCR and Sequencing Workflows

Streamlining of PCR and Sequencing Workflows. Natalie Morrell West Midlands Regional Genetics Laboratory. Prior to Streamlining of PCR Set up. Automated PCR setup BRCA HNPCC FAP Gorlins SOTOS CHARGE Using primer spotted plates

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Streamlining of PCR and Sequencing Workflows

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  1. Streamlining of PCR and Sequencing Workflows Natalie Morrell West Midlands Regional Genetics Laboratory

  2. Prior to Streamlining of PCR Set up Automated PCR setup • BRCA • HNPCC • FAP • Gorlins • SOTOS • CHARGE • Using primer spotted plates • Requires a set number of patient samples (typically 2 - 4 patients) Manual PCR setup • Prenatals • Presymptomatics • Confirmations • All other full gene screens • Repeat PCRs following an initial screen • PCR reactions set up in 0.5ml tubes by GTs and Scientists • Very labour intensive and repetitive process

  3. Problems to overcome prior to Automation of Manual PCRs • PCR reactions for different genes/amplicons optimised using: • different PCR cycling conditions • Different types of Taq • Different reaction volumes • Variable number of amplicons for each gene (from 2 to 38) • Variable number of patients to be screened for each gene / amplicon

  4. Touchdown PCR Conditions • 95°C for 5 mins • 95°C for 1 min • 65-55°C for 1 min • AT decreases by 0.5°C each cycle • 72°C for 1 min • 95°C for 1 min • 55°C for 1 min • 72°C for 1 min • 72°C for 10 mins 20 cycles 10 cycles

  5. Diseases (Genes) Tested • BHD (FLCN) • BRCA (BRCA 1 & 2) • FAP (APC) • GACA (CDH1) • HNPCC (MLH1, MSH2 & MSH6) • HPRC (MET) • MEN2 (RET) • MAP (MYH) • PP (SDHB, SDHC & SDHD) • VHL (VHL) • ALSTROM (ALMS) • ARC (VPS33b) • CMT (GJB1, MPZ & PMP22) • Deafness (CX26) • DIDMOAD (WFS1) • INAD (PLA2G6) • MICRO Syndrome (RAB3GAP1) • MPS (CHRNG) • PHP (PIT1 & PROP1)

  6. Gel Photos 55°C HNPCC (MLH1) 57°C FAP 58/61°CDIDMOAD 61°C INAD 60/64°CBHD 60°C CMT (MPZ) 63°C MEN2 63°C ALSTROM

  7. Touchdown PCR Test Results • 348 out of 376 (92.6%) amplicons produced a PCR product suitable for sequencing • 19 out of the 28 amplicons which failed to amplify were for FAP or HNPCC • primers originally designed for dHPLC • Another 6 of the amplicons were notoriously temperamental

  8. Problems to overcome prior to Automation of Manual PCRs • PCR reactions for different genes/amplicons optimised using: • different PCR cycling conditions • Different reaction volumes • Different types of Taq (MegaMix Original, MegaMix W or AmpliTaq Gold) • Variable number of amplicons for each gene (from 2 to 38) • Variable number of patients to be screened for each gene / amplicon

  9. Creation of an Excel Template

  10. Automated PCR Systems Full Gene Screens • Introduced in July 2009 • Robot transfers primers followed by a PCR mix containing DNA and MegaMix into a 96 well plate • Automated PCR set up now used for almost all full gene screens currently done in lab • Gene Compatible 47 • Amplicons 640 • Total Gene screens 1862 • Total PCRs set up 22185

  11. Total Number of Automated Full Gene ScreensJuly 09-Oct 10

  12. Total Number of Automated PCR Reactions July 09-Oct 10

  13. Automated PCR Systems Full Gene Screens Non Gene Screen Introduced in March 2010 Automated PCR set up of Presymptomatics and confirmations PCRs repeated from Automated Gene Screens Robot transfers the DNAs, Primers and Megamix separately into a 96 well plate Amplicons 759 Total PCRs 5184 • Introduced in July 2009 • Robot transfers primers followed by a PCR mix containing DNA and MegaMix into a 96 well plate • Automated PCR set up now used for almost all full gene screens currently done in lab • Gene Compatible 47 • Amplicons 640 • Total Gene screens 1862 • Total PCRs set up 22185

  14. E-Gels • Pre made agarose gels • Ethidium Bromide stained • Gels completely enclosed in plastic • Can be used on robots for automated gel loading from 96 well plates

  15. E - Gels Typical e-gel photo Patient Samples Negative Controls

  16. Sequencing Workflow Automated PCR Plates (BRCA, Gene Screen and non Gene Screen) Manual PCRs Post PCR Clean up Sequencing Reaction Setup Post Sequencing Reaction Clean up Semi Automated Capillary Electrophoresis Sequence Traces are analysed

  17. Not currently automated • Prenatal work • Nested PCRs e.g. CAH • RNA studies • Tumour work • DNA extracted from paraffin blocks • HNPCC (PMS2) • due to pseudogene • Non diagnostic work (Validation/ Primer optimisation) • Molecular Pathology work • DNA extracted from paraffin blocks

  18. PCRs set up July – Sept 2010 • TOTAL PCRs 18343 • Automated Set up16681 • Primer spotted plates (BRCA) 8316 • Automated Full Gene screen 6061 • Automated Non Gene screen 2304 • Manual Set up1662 • Diagnostic 901 • Validation 366 • Molecular Pathology 395 94.8% of diagnostic PCR setups performed by the Molecular Genetics lab are now automated

  19. Benefits of a Streamlined Process • Less ‘hands on’ time in lab • Faster turnaround times due to less sample batching • More consistent results (95.8% of PCRs work 1st time) • Reduced risk of contamination • has led to reduction in number of negative controls used on Gene Screen PCR plates • Higher level of consistency in PCR set up highlights problematic primers faster Saves money! • More streamlined process “easier” to integrate into StarLIMS

  20. Acknowlegements • Development of new screening strategy • Yvonne Wallis • Hayley Simm • GTs involved in the lab work • Anna Yeung • Mohammed ZishanIqbal • Reena Patel • Sam Court • CarlyMogg • Gemma Andrews

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