1. Nuts and Bolts of Clinical Genomic Sequencing Thomas Stricker MD PhD Vanderbilt University

2. Next Generation Sequencing Illumina Seqeuncing Technology DNA – the genetic code DNA is a double stranded polymer of 4 bases (A, T, C,G) The order (sequence) of A,T,C,G is the genetic code A always pairs with T on the opposite strand, and C always pairs with G Enzymes called polymerases make copies of DNA by taking a single strand of DNA, and then adding A,T,C,G according to the base-pairing rules Sanger (mod by Lee Hood) Sequencing by synthesis Mix many copies of the same DNA molecule, polymerase, ATCGs, and a small amount of flourescently labeled ATCG that are terminated Terminated bases stop extension Separate based on size

3. 1. In vitro amplification, ‘cloning’ 2. Flow cell based sequencing by synthesis 3. A draft of the human genome Illumina Seqeuncing Technology What Happened?

4. Illumina Seqeuncing Technology

5. Single platform – 4 mutation types

6. 877 Lung Specimens (843 patients) 7/1/2010-2/28/2013 * * Data courtesy of Dr. William Pao and Dr. Mia Levy OncogeneFrequency (%) Treatment EGFR10-35Gefitinib, erlotinib, afatinib ALK fusion3-7Crizotinib MET amp2-4Crizotinib DDR2~4Dasatinib HER22-4Afatinib ROS1 fusion1Crizotinib BRAF Y472CrareDasatinib BRAF V600E1Vemurafenib, dabrafenib RET fusion1Cabozantinib NRAS1Trametinib (preclinical) KRAS15-25Selumetinib (with chemo) FGFR1/2 amp ~20AZD4547 Broad spectrum of mutations gives physicians some information… …but without well-annotated sequencing reports, physicians struggle to find best therapy Rare Mutations – Implications for Therapy

7. The Long Tail of Cancer mutations 45 Recurrently mutated genes in the TCGA breast cancer data set Range from over 30% to 2% of cases

8. Human Genome 3 billion base pairs in the human genome Roughly 1% is in coding sequence

9. Target Enrichment = Amplicon-based approach

10. Target Enrichment = Hybrid capture approach

11. Fusion Detection = Hybrid Capture

12. Tumor-Normal Contamination

14. Clinical Utility of NGS

17. Analysis Schematic

18. MS Lawrence et al. Nature 000, 1-5 (2013) doi:10.1038/nature12213. Somatic mutation frequencies observed in exomes from 3,083 tumour–normal pairs

19. Inherited Variants > Somatic Synonymous SNVs Non-Synonymous SNV

20. Inherited Variants > Somatic Somatic Coding mutations – 3 to 300

21. IGV – Genotyping