Delivering a Pharmacogenomics NGS service in 5 working

Delivering a Pharmacogenomics NGS service in 5 working days Jonathan Edgerley Genetic Technologist

Pharmacogenomics • The study of the role of genetics in drug response. A bridge towards ‘personalised medicine’ • In oncology, tumour tissue biopsy and the development of circulating tumour DNA (ct. DNA) sequencing allows us to identify somatic variants in oncogenes whose affects can be inhibited by targeted drug therapy • Genes such as EGFR and ALK in lung cancer, KRAS and NRAS in colorectal cancer and BRAF in Melanoma. Mutations in each can lead to over activation of their related cell-signalling pathways, resulting in uncontrolled cell proliferation • A quick and accurate diagnosis of these changes can determine the correct drug and dosage required as treatment to arrest the effects of this over activation in order to prolong a patients life • “We are seeing a trend emerge with more cancers with genetic classification. Genomics is helping us to define the disease based on the genetic markers that may drive the malignancy and may serve as therapeutic targets. ” – John Leite, VP of Marketing for Illumina’s Oncology business

Aim of the service • Samples of extreme urgency, DNA extracted from tumour cells – often minimal quantity and poor quality of DNA (FFPE) • Require a fast, high throughput protocol which enables massively parallel sequencing of such DNA with no compromise on sequencing data quality or coverage • Require improved sequencing coverage and enhanced detection of a greater number of clinically actionable variants from multiple oncogenes • A test that caters for all our Pharmacogenomic cancer referrals, potentially purging all current testing strategies into a single workflow which provides an innovative, in-depth and wide-scoping tumour Next-Generation Sequencing service • All to be delivered within a 5 working day TAT

What did we arrive at? • A custom workflow which brings together a Qiagen Target Enrichment based amplification kit and an Illumina Library Preparation kit • Qiagen Gene. Read DNAseq Targeted Panels V. 2 and Gene. Read DNAseq Panel PCR Kit V. 2 • Illumina Tru. Seq DNA PCRFree Library Preparation Kit

Workflow Overview Pre. Analytical • DNA Extraction • Sample Qubit Quantification and DNA Dilution Panelling Sample Panelling and Worksheet Preparation Amplification Qiagen Gene. Read PCR • Gene. Read Post-PCR Gel QC Panel Pooling Master Mix CP Pooling Library Prep Adenylate 3’ Ends and Adaptor Ligation Final Library Purification Tru. Seq Final Libraries Purification Size Selection Purification Final Library Quantificatio n Gene. Read Size Selection AMPure Purification Final Libraries q. PCR Quantification Dilution and End Repair Pooling Libraries Tapestation Quantification and Size Selection QC • Gene. Read Dilution (100 ng/60 ul) and Illumina Tru. Seq End Repair PAL Tru. Seq Purification Loading Libraries Tru. Seq Sample Purification Mi. Seq Loading and Sequencing

Amplification Qiagen Gene. Read DNAseq Targeted Panel V. 2 • • Human Clinically Relevant Tumor Panel – 24 genes Human BRCA 1 and BRCA 2 Panel Multiplex PCR-enabled target enrichment of genomic regions of interest As little as 10 ng DNA needed (aim for 20 ng per Primer Pool) Compatible with many samples types including FFPE samples Employs overlapping primer sets across the exonic portions of a gene or group of genes to maximize target coverage Primer sets divided into 4 Primer Pools (Master Mixes) to maximize specificity. Following amplification, enriched regions from each sample are pooled together, yielding one library preparation for each sample.

Gel Electrophoresis

Gene. Read Size Selection Purification

Library Preparation Illumina Tru. Seq® DNA PCRFree Sample Preparation Kit • Well defined and widely adopted Library Preparation chemistry • Elimination of PCR amplification and replacement of gel-based size selection with bead-based selection significantly streamlines the workflow • Elimination of PCR amplification steps removes typical PCR-induced library bias and coverage gaps • Excellent data quality and detailed sequencing information for challenging regions of the genome • Results in a fast, high throughput protocol with exceptional data quality and improved coverage uniformity

How fast? Pre. Analytical • DNA Extraction • Sample Qubit Quantification and DNA Dilution Panelling Sample Panelling and Worksheet Preparation Amplification Qiagen Gene. Read PCR • Gene. Read Post-PCR Gel QC Panel Pooling Master Mix CP Pooling Library Prep Adenylate 3’ Ends and Adaptor Ligation Final Library Purification Tru. Seq Final Libraries Purification Size Selection Purification Final Library Quantificatio n Gene. Read Size Selection AMPure Purification Final Libraries q. PCR Quantification Dilution and End Repair Pooling Libraries Tapestation Quantification and Size Selection QC • Gene. Read Dilution (100 ng/60 ul) and Illumina Tru. Seq End Repair PAL Tru. Seq Purification Loading Libraries Tru. Seq Sample Purification Mi. Seq Loading and Sequencing

Automation Pre. Analytical • DNA Extraction • Sample Qubit Quantification and DNA Dilution Panelling Sample Panelling and Worksheet Preparation Gene. Read Amplification Qiagen Gene. Read PCR • Gene. Read Post-PCR Gel QC Panel Pooling Master Mix CP Pooling Tru. Seq Library Prep Adenylate 3’ Ends and Adaptor Ligation Final Library Purification Tru. Seq Final Libraries Purification Size Selection Purification Final Library Quantificatio n Gene. Read Size Selection AMPure Purification Final Libraries q. PCR Quantification Dilution and End Repair Pooling Libraries Tapestation Quantification and Size Selection QC • Gene. Read Dilution (100 ng/60 ul) and Illumina Tru. Seq End Repair PAL Tru. Seq Purification Loading Libraries Tru. Seq Sample Purification Mi. Seq Loading and Sequencing

Automation • A custom ‘hybrid’ workflow between Qiagen and Illumina kits offers no ‘off the shelf’ automation capacity • Using Beckman Coulter Biomek NX Span-8 systems, ‘in-house’ customised programming has enabled tailoring automation to the workflow • As such, effectively adapted existing technology to design a semiautomated workflow for the Qiagen Gene. Read DNAseq PCR and Illumina Tru. Seq PCR-free Library Preparation • A workflow with increased capacity, scalability, accuracy, and reliability(? ) • Crucially, allows valuable ‘walk-off’ time to manage numerous panels simultaneously

Finally… No confirmations!

Acknowledgments • Dr. Andrew Wallace. Consultant Clinical Scientist • Dr. George Burghel and Ronnie Wright. Clinical Scientist • John Dunlop. Technical Development Manager • Laura Dutton. Senior Genetic Technologist • Pre-Analytical Team.