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 (ctDNA) 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 GeneRead DNAseq Targeted Panels V.2 and GeneRead DNAseq Panel PCR Kit V.2 Illumina TruSeq DNA PCR-Free Library Preparation Kit

Workflow Overview DNA Extraction Sample Qubit Quantification and DNA Dilution Pre-Analytical Sample Panelling and Worksheet Preparation Panelling Qiagen GeneRead PCR Amplification Gel QC Post-PCR Master Mix CP Pooling Panel Pooling GeneRead Size Selection AMPure Purification Size Selection Purification GeneRead Tapestation Quantification and Size Selection QC GeneRead Dilution (100ng/60ul) and Illumina TruSeq End Repair Dilution and End Repair TruSeq Sample Purification TruSeq Purification Adenylate 3’ Ends and Adaptor Ligation Library Prep TruSeq Final Libraries Purification Final Library Purification Final Libraries qPCR Quantification Final Library Quantification PAL Pooling Libraries MiSeq Loading and Sequencing Loading Libraries

Amplification Qiagen GeneRead DNAseq Targeted Panel V.2 Human Clinically Relevant Tumor Panel – 24 genes Human BRCA1 and BRCA2 Panel Multiplex PCR-enabled target enrichment of genomic regions of interest As little as 10ng DNA needed (aim for 20ng 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. 98.1% coverage for Clinically Relevant Tumor panel. 100% coverage for BRCA1 and BRCA2.

Gel Electrophoresis

GeneRead Size Selection Purification Both samples shared similar percentage portions of the indexing reads. How?...TruSeq. – 100ng/60ul. 99.6% at 30x and 97.6% at 100x 98.5% at 30x and 96.6% at 100x

Library Preparation Illumina TruSeq® DNA PCR-Free 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? DNA Extraction Sample Qubit Quantification and DNA Dilution Pre-Analytical Sample Panelling and Worksheet Preparation Panelling Qiagen GeneRead PCR Amplification Gel QC Post-PCR Master Mix CP Pooling Panel Pooling GeneRead Size Selection AMPure Purification Size Selection Purification GeneRead Tapestation Quantification and Size Selection QC GeneRead Dilution (100ng/60ul) and Illumina TruSeq End Repair Dilution and End Repair TruSeq Sample Purification TruSeq Purification Adenylate 3’ Ends and Adaptor Ligation Library Prep TruSeq Final Libraries Purification Final Library Purification Final Libraries qPCR Quantification Final Library Quantification PAL Pooling Libraries MiSeq Loading and Sequencing Loading Libraries Potential need to process 3 panels a week (9 days work in 5 days). With elimination of PCR amplification steps, little ‘walk-off’ time to operate multiple panels at differing stages of the workflow. There are, however, lots of purification steps that are long, repetitive and time consuming when done manually. But not when automated…

Automation DNA Extraction Sample Qubit Quantification and DNA Dilution Pre-Analytical Sample Panelling and Worksheet Preparation Panelling Qiagen GeneRead PCR GeneRead Amplification Gel QC Post-PCR Master Mix CP Pooling Panel Pooling GeneRead Size Selection AMPure Purification Size Selection Purification GeneRead Tapestation Quantification and Size Selection QC GeneRead Dilution (100ng/60ul) and Illumina TruSeq End Repair Dilution and End Repair TruSeq Sample Purification TruSeq Purification Adenylate 3’ Ends and Adaptor Ligation TruSeq Library Prep TruSeq Final Libraries Purification Final Library Purification Final Libraries qPCR Quantification Final Library Quantification PAL Pooling Libraries MiSeq Loading and Sequencing Loading Libraries

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 semi-automated workflow for the Qiagen GeneRead DNAseq PCR and Illumina TruSeq 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.