Nuts and Bolts of Clinical Genomic Sequencing

Thomas Stricker MD PhDVanderbilt University

Next Generation SequencingIllumina Seqeuncing TechnologyDNA – 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 *

No mutation detected

(512)56.1%

EGFR (135)14.8%

BRAF (20)2.2%

AKT1 (2)0.2%

PTEN (2)0.2%

PIK3CA (20)2.2%

NRAS (5)0.5%

MEK1 (8)0.9%

KRAS (198)21.7%

ERBB2 (10)1.1%

* Data courtesy of Dr. William Pao and Dr. Mia Levy

Oncogene Frequency (%)

Treatment

EGFR 10-35 Gefitinib, erlotinib, afatinib

ALK fusion 3-7 Crizotinib

MET amp 2-4 Crizotinib

DDR2 ~4 Dasatinib

HER2 2-4 Afatinib

ROS1 fusion 1 Crizotinib

BRAF Y472C rare Dasatinib

BRAF V600E 1 Vemurafenib, dabrafenib

RET fusion 1 Cabozantinib

NRAS 1 Trametinib (preclinical)

KRAS 15-25 Selumetinib (with chemo)

FGFR1/2 amp

~20 AZD4547

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

Tumor-Normal Contamination

Clinical Utility of NGS

Clinical Utility of NGS

Clinical Utility of NGS

Analysis Schematic

MS Lawrence et al. Nature 000, 1-5 (2013) doi:10.1038/nature12213

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Somatic mutation frequencies observed in exomes from 3,083tumour–normal pairs

Inherited Variants > Somatic

Synonymous SNVsNon-Synonymous SNV

Inherited Variants > Somatic

Somatic Coding mutations – 3 to 300

IGV – Genotyping