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
.
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
Top Related