1. Exceptional healthcare, personally delivered www.nbt.nhs.uk/genetics Prenatal arrays: The BGL experience 20 th November 2014 Rob Moore Genetic Technologist

2. Exceptional healthcare, personally delivered Contents EACH at BGL ­ EACH samples ­ DNA extractions ­ EACH aCGH Prenatal array development ­Extractions ­Processing throughput ­Processing time http://www.nbt.nhs.uk/genetics

3. Exceptional healthcare, personally delivered EACH Evaluation of Array Comparative Genomic Hybridisation in Prenatal Diagnosis of Foetal Anomalies. “Evaluate the efficacy of prenatal array CGH and address the hypothesis that array CGH detects more de novo pathogenic chromosomal imbalances than standard karyotyping” AF, CVS & FB samples from pregnancies with positive ab-scans and/or NT > 3.5mm. In total 186 EACH samples ­ 79 AF, 105 CVS, 2 FB 127 proceeding to aCGH ­68 AF, 55 CVS, 2 FB http://www.nbt.nhs.uk/genetics

4. Exceptional healthcare, personally delivered EACH sample work flow http://www.nbt.nhs.uk/genetics EACH & parental bloods CVS Total sample ≥ 10mg proceed on uncultured CVS EACH CVS extraction If < 10mg then sample is cultured Manual Cultured Cell extraction AF Total sample ≥ 18ml proceed on uncultured AF iGENatal extraction If < 18ml then sample is cultured Manual Cultured Cell extraction FB Gentra or QFPCR DNA if suitable QFPCR EZ1/Gentra If DNA unsuitable sample is cultured Manual Cultured Cell extraction

5. Exceptional healthcare, personally delivered EACH Extractions EACH CVS: 2.5 – 4mg of sample Lab developed manual protocol Labour intensive and time consuming. Overnight incubation step + a further half day of lab work. Produces high quality un-sheared DNA. iGENatal: 2 – 3ml AF + 10ml supernatant iGEN Biotech phenol based extraction kit Labour intensive, uses hazardous chemicals. Produces un-sheared DNA, varied levels of phenol contamination. Quantified by Qubit due to phenol contamination. Manual Cultured Cell : nunc tube culture Same protocol as EACH CVS, reduced incubation (30mins) http://www.nbt.nhs.uk/genetics

6. Exceptional healthcare, personally delivered EACH arrays Quantity of DNA determined type of array processing. Standard array processing 500ng total DNA loaded onto array (Agilent 8x60k oligo array). Identical to current postnatal array processing. Reduced array processing 150ng total DNA loaded onto array (Agilent 8x60k oligo array). Requires a custom reference in order to be processed. Parental follow up arrays Identical to current PvP array processing. http://www.nbt.nhs.uk/genetics

7. Exceptional healthcare, personally delivered Extractions Current EACH extraction protocols are too slow/too labour- intensive. Extractions need to be streamlined and automated. Pooling QFPCR and aCGH samples for QFPCR EZ1 extraction ~ 5ml AF or ~3mg CVS to be run on EZ1 tissue protocol. Prenatal array development http://www.nbt.nhs.uk/genetics

8. Exceptional healthcare, personally delivered Prenatal array development Processing throughput Current aCGH ~50 samples per week. Processing runs of 24 samples twice a week. Future sample numbers 5+ Prenatal samples a week (highly variable) 20 - 30 solid tissue samples a week ?20 NGS confirmation array samples a week HT processing 48 sample processing Increased automation/robot time ­ Dilutions, PCR set up, sample/ref combination http://www.nbt.nhs.uk/genetics

9. Exceptional healthcare, personally delivered Prenatal array development Sample processing time Current 3 day aCGH processing Potential 2 day aCGH processing http://www.nbt.nhs.uk/genetics Day 1 Sample labelling Day 2 Array hybridisation Day 3 Array washing Day 1 Sample & Array labelling hybridisation Day 2 Array washing

10. Exceptional healthcare, personally delivered http://www.nbt.nhs.uk/genetics Acknowledgements A big thank you to all BGL staff (past and present) for all your help and for making the EACH project bearable…..roll on prenatal arrays