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

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

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

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

Illumina Seqeuncing Technology

Single platform – 4 mutation types

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

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

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

Target Enrichment = Amplicon-based approach

Target Enrichment = Hybrid capture approach

Fusion Detection = Hybrid Capture

Tumor-Normal Contamination

Clinical Utility of NGS

Analysis Schematic

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

Inherited Variants > Somatic Synonymous SNVs Non-Synonymous SNV

Inherited Variants > Somatic Somatic Coding mutations – 3 to 300

IGV – Genotyping