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