advanced ngs data analysis & interpretation- bgw + iva: ngs tech overview webinar series part 3
TRANSCRIPT
Sample to Insight
1
Mutational analysis using QIAGEN’s GeneRead panels and Sample-to-Insight NGS solutions
Raed Samara, PhD
Part 3Data AnalysisInterpretation
Sample to Insight
NGS part 3: Targeted NGS for Cancer Research 2
Legal disclaimer
• QIAGEN products shown here are intended for molecular biology
applications. These products are not intended for the diagnosis,
prevention or treatment of a disease.
• For up-to-date licensing information and product-specific
disclaimers, see the respective QIAGEN kit handbook or user
manual. QIAGEN kit handbooks and user manuals are available
at www.QIAGEN.com or can be requested from QIAGEN
Technical Services or your local distributor.
Sample to Insight
Precision medicine: right drug, right patient, right time and dose
‘One size fits all’ does not work
NGS part 3: Targeted NGS for Cancer Research 3
Sample to Insight
Mutations
AGCTCGTTGCTCAGCTCReference genome
AGCTCGTTGCTCAGCGTTCInsertion
AGCTC---GCTCAGCTC
Deletion
Indels Copy number variations
T
G
CA
T GA
C
DNA variants for precision medicine
RS NGS part 3: Targeted NGS for Cancer Research 4
Sample to Insight
Mutations
AGCTCGTTGCTCAGCTCReference genome
AGCTCGTTGCTCAGCGTTCInsertion
AGCTC---GCTCAGCTC
Deletion
Indels Copy number variations
Actionable DNA Variant BRAF V600E EGFR E746-750
+ Kinase domain mutation HER2
Disease Melanoma Lung adenocarcinomas IDC-Breast cancer
Therapy Vemurafenib (PLX4032) Erlotinib/ Gefitinib Trastuzumab
Actionable DNA variants for precision medicine
Only a handful of mutations are actionable
RS NGS part 3: Targeted NGS for Cancer Research 5
Sample to Insight
Actionable DNA variants for precision medicine
How many?
RS NGS part 3: Targeted NGS for Cancer Research 6
Sample to Insight
• How many mutations to test for?• How to test for these mutations?
o Sequential testingo Parallel testing
EGFR (L858R)
KRAS (G12C)
+
Response rates of >70% in patients with
non-small cell lung cancer treated with either erlotinib or
gefitinib
Poor response rate in patients with non-small
cell lung cancer treated with either erlotinib or gefitinib
Adapted from Govindan et al., Cell 150 (2012)
Precision medicine for lung cancer
Current lung cancer biomarker landscape
KRAS25%
EGFR
23%
EML4-ALK6%
BRAF3%
PIKC3A3%
MET2%
ERBB2
1%
MAP2K10.4%
NRAS
0.2%
Un-know
n37%
RS NGS part 3: Targeted NGS for Cancer Research 7
Sample to Insight
Actionable DNA variants for precision medicine
Targeted DNA sequencing delivers accurate information required for precision medicine
Attribute/Parameter
Information level
Cost per sample
Coverage achieved
DNA input
No. of samples multiplexed
Whole Genome
Sequencing
3 x 109 bps
$5000
30x
1 µg
1^
Whole Exome
Sequencing
5 x 107 bps
$2000
100x
100 – 200 ng
2*
Targeted DNA
Sequencing
6 x 104 bps#
$200
1000x
10 ng
96*
Benefits of Targeted DNA Sequencing:
Clinical utility requires targeted analysis
More relevant data
More cost effective
Detect low-frequency mutations
Lower DNA requirements
Higher multiplexing capabilities
RS NGS part 3: Targeted NGS for Cancer Research 8
Sample to Insight
Why choose targeted DNA sequencing?
• Well-defined content
• Small target size
• More reads per sample
• Examine variants that matter
• Multiplex many samples to save money
• Detect low frequency variants
Features Benefits
Targeted DNA sequencing limits the genes or targets to be sequenced
RS NGS part 3: Targeted NGS for Cancer Research 9
Sample to Insight
Targeted DNA sequencing (TDS)
Shrink the genome
Sample Insight
The principle of targeted enrichment is to sequence millions of small DNA fragments that represent the region of interest, simultaneously
gDNAVariants Report
KRAS G12DEGFR T790MIDH1 R132H
KRASEGFRIDH1
RS NGS part 3: Targeted NGS for Cancer Research 10
Sample isolation
Targeted enrichment
Library construction NGS run Data
analysis Interpretation
Sample to Insight
NGS part 3: Targeted NGS for Cancer Research
Sample-to-Insight: Integrated universal targeted NGS workflow
11
To overcome NGS challenges
Sample Insight
Turnaround time, and limited
amounts of DNA
Library yield Coming soon
Data processing & Variant calling
Isolation of high-quality DNA
samples
Quantifying amplifiable (not
total) amounts of DNA
Clinical & Biological
interpretation of data
RS
Sample isolation
Targeted enrichment
Library construction NGS run Data
analysis Interpretation
Sample QC Library QC
Variantconfirmation
Sample to Insight
Why choose multiplex PCR-based targeted enrichment?
• Offers specificity that beats capture-based approaches
• Offers uniformity that beats capture-based approaches
• Use sequencing capacity on regions targeted by the panel, with minimal off-target sequencing
• Achieve more uniform enrichment for more sequencing efficiency
Features Benefits
It delivers unmatched specificity and uniformity (compared to capture-based methods)
RS NGS part 3: Targeted NGS for Cancer Research 12
Sample to Insight
GeneRead DNAseq Panels V2
Multiplex PCR-based enrichment of gene(s) or genomic region(s)
Builds on our 10-year experience in designing PCR assays
GeneRead DNAseq Panel V2 GeneRead DNAseq PCR Kit V2
Collection of primer pairs dispended into primer pools/tubes. Primers correspond to the targeted
region(s) (Gene 1 and Gene 2, as example)
Primer pair; amplicon
RS NGS part 3: Targeted NGS for Cancer Research 13
Sample to Insight
Gene 1 Gene 2
Primer design algorithm
GeneRead DNAseq Panels V2
Multiplex PCR-based enrichment of gene(s) or genomic region(s)
PCR Chemistry
Primer separation algorithm
GeneRead DNAseq Panel V2(sets of primer pools/tubes)
GeneRead DNAseq PCR Kit V2
RS NGS part 3: Targeted NGS for Cancer Research 14
Sample to Insight
GeneRead DNAseq Panels V2
Product description
Sample Insight
ACCAGTGAC
TATAGCTAG
GTCCTATTG
CCGGTGTAC
Variants Report
Ref: AAGTCTCase: AACTCT
GeneRead DNAseq Panel V2
GeneRead DNAseq PCR Kit V2
DNA
Amplicons
Library
Reads
GeneRead DNAseq panels enrich genomic targets by generating amplicons (small ds DNA fragments) through PCR
RS NGS part 3: Targeted NGS for Cancer Research 15
Sample isolation
Targeted enrichment
Library construction NGS run Data
analysis Interpretation
Sample to Insight
Clinically- and biologically-focused panels
Cataloged panels; bench-tested Type Panel name
Solid TumorClinically Relevant Tumor
Tumor Actionable Mutations
Hematologic malignancies Myeloid Neoplasms
Tissue Specific
Breast Cancer
Colorectal Cancer
Liver Cancer
Lung Cancer
Ovarian Cancer
Prostate Cancer
Gastric Cancer
Cardiomyopathy
Gene(s)-specific BRCA1 and BRCA2
Comprehensive
Cancer Predisposition
Comprehensive Cancer
Carrier testing
Cancer Genome Census
ClinVar
Largest collection of pre-designed panels suitable for a wide range of needs
RS NGS part 3: Targeted NGS for Cancer Research 16
Sample to Insight
Application-specific amplicon design
Builds on >10 years in assay development
G12
Deep (redundant) tiling amplicon designDense overlap amplicon design
Sparse overlap amplicon design
Primers Amplicons
RS NGS part 3: Targeted NGS for Cancer Research 17
Sample to Insight
Customized panels
Custom and Mix-n-Match
1 No competitor offers bench-tested gene designs2 Fastest on the market
What is the list of your targets?
RS NGS part 3: Targeted NGS for Cancer Research 18
GeneRead Mix-n-Match
GeneReadCustom
• Access to 570 bench-tested gene designs1
• Fixed specifications• Turnaround time = 3 days2
• Bioinformatically target any gene(s) or genomic region(s) within the human genome
• Flexible specifications• Turnaround time = 14 days2
Sample to Insight
The power of Mix-n-Match pool of validated genes
Consistent performance00
4X00
8X01
3X50
1X00
2X00
3X00
6X50
1X00
6X01
3X50
1X00
1X00
2X00
4X00
6X00
7X00
8X01
3X50
1X00
1X00
6X00
7X01
3X50
1X00
1X00
3X00
6X00
7X01
3X50
1X00
1X00
8X01
3X50
1X00
1X00
2X00
3X00
4X00
6X00
7X00
8X01
3X50
1X
0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
70.0%
80.0%
90.0%
100.0%
Experimental Coverage UniformityMET NRAS TP53PDGFRA PIK3CA PTEN RB1 RET
RS NGS part 3: Targeted NGS for Cancer Research 19
Sample to Insight
Why choose GeneRead panels workflow for targeted enrichment?
• Small target sizes
• Uses Multiplex PCR
• Achieve a high degree of coverage depth
• Low DNA input material
• Quick turnaround time
• No physical removal of primer sequences
• Platform-independence
• Pool amplicons before library construction
• Integrated workflow
• Multiplex many samples to save money
• Achieve higher specificity and uniformity than other approaches1
• Make more confident calls to detect low frequency variants 4
• Sequence low-yielding samples such as FFPE and cfDNA samples
• Go from sample to insight quickly
• Reduce false positives and negatives2
• Achieve 100% specificity and 100% sensitivity3
• Use one panel for your different sequencers
• Generate one library per sample to save money
• Sequence low-quality samples
Features Benefits
GeneRead DNAseq panels address many NGS challenges
1. Mamanova et al. 2010. Nature Methods, 7(2):111 2. Satya and DiCarlo. 2014. BMC Genomics, 15:10733. Heydt et al. 2015. BMC Cancer, 15:291 4. Helen Fernandes, AMP 2014
RS NGS part 3: Targeted NGS for Cancer Research 20
Sample to Insight
Why choose GeneRead panels workflow for targeted enrichment?
Achieve better variant calling accuracy to reduce false negatives
RS NGS part 3: Targeted NGS for Cancer Research 21
Sample to Insight
Why choose GeneRead panels workflow for targeted enrichment?
Achieve 100% specificity and 100% sensitivity through better variant calling accuracy
The sensitivity of the AmpliSeq panel on the MiSeq™ (Illumina) was only 93%. Using this panel, four of the exon 11 mutations (p.M552_K558del, p.M552_V559del, K550_K558del, c.1648-5_1672del) could not be detected as these mutations were at the amplicon boundaries and primer binding sites.
RS NGS part 3: Targeted NGS for Cancer Research 22
Sample to Insight
Why choose GeneRead panels workflow for targeted enrichment?
Integrated workflow to optimally-sequence low-quality samples
The GeneRead workflow enables the optimal sequencing of low-yielding and low quality FFPE samples
Data presented by Helen Fernandes (Cornell University) at AMP 2014
AmpliSeq workflow GeneRead workflow
Group # of samples
DNA Quant Library quality & quantity DNA Quality Library quality &
quantity
Qubit Agilent QuantiMIZE qPCR
1 30
2 3
3 6 X
XSequenced optimallyNot sequenced optimally
RS NGS part 3: Targeted NGS for Cancer Research 23
3
Sample to Insight
Compatibility with major sequencing platforms
Same panel for different platforms
IlluminaQIAGEN
Ion
GeneRead® DNAseq Panels V2
HiSeq 2500MiSeq
NextSeq
PGMProton
Confirm variants on different platforms using the same panel
RS NGS part 3: Targeted NGS for Cancer Research 24
Sample to Insight
Sample-to-Insight: Integrated universal targeted NGS workflow
To overcome NGS challenges
Sample Insight
Turnaround time, and limited
amounts of DNA
Library yield Coming soon
Data processing & Variant calling
Isolation of high-quality DNA
samples
Quantifying amplifiable (not
total) amounts of DNA
Clinical & Biological
interpretation of data
RS NGS part 3: Targeted NGS for Cancer Research 25
1
Sample isolation
Targeted enrichment
Library construction NGS run Data
analysis Interpretation
Sample QC Library QC
Variantconfirmation
Sample to Insight
Sample-to-Insight: Integrated universal targeted NGS workflow
Bioinformatics solutions
Sample
Insight
Upstream Analysis ‘Primary’ ‘Secondary’ ‘Tertiary’
NGS part 3: Targeted NGS for Cancer Research 26
Sample Prep
AssayData
Sequence-Level
Statistics
Biology of Interest(Genes,Variants,
etc.)
Annotation & Comparative(Statistical Analysis)
Annotation & Biological
Interpretation
Sample to Insight
CLC Biomedical: Flexible workflows tailored for end-users
Lock key parameters of workflow to standardize processing
Identify Variants Example
Sample Prep
Plug-in for GeneRead DNAseq targeted panels
NGS part 3: Targeted NGS for Cancer Research 27
Assay Data
Sequence- Level
Statistics
Biology of Interest
Annotation & Comparative
Analysis
Annotation & Comparative Interpretation
Sample to Insight
IVA: Rapid prioritisation and annotation of variants
Examine a single sample or compare multiple samples
NGS part 3: Targeted NGS for Cancer Research 28
Sample to Insight
Examine evidence and references
• Variant Findings• ACMG Classification Rules• Variant Views
NGS part 3: Targeted NGS for Cancer Research 29
Sample to Insight
Why choose targeted DNA sequencing?
Detect known & discover novel variants
AM & CR
Disease-specific Comprehensive
DetectionDiscovery
MultiplexingTarget size
Custom & Mix-n-Match
Pan
els
App
licat
ions
Spe
cific
atio
ns
Clinical research Translational & Discovery research
Whole Exome SeqWhole Genome Seq
Targeted DNA sequencing: robust detection, limited discoveryAM: actionable mutations panel; CR: clinically relevant panel
RS NGS part 3: Targeted NGS for Cancer Research 30
Sample to Insight
Novel mutation discovery by targeted sequencing
Gene panels can facilitate the discovery of novel mutations
Notably, this study facilitated the identification of BRCA2 Thr9976, which is the strongest genetic association in lung cancer reported so far. For a smoker carrying this variant (2% of the population), the risk of developing lung cancer is approximately doubled, which may have implications for identifying high-risk ever-smoking subjects for lung cancer screening. Additionally, future study of the effects of PARP inhibition in smokers with lung cancer carrying BRCA2 Thr9976 may be warranted.
RS NGS part 3: Targeted NGS for Cancer Research 31
Sample to Insight
Clinical value of targeted sequencing with targeted panels
More is not necessarily better; in some cases, less is better
RS NGS part 3: Targeted NGS for Cancer Research 32
Sample to Insight
GeneRead Targeted Panels for NGS: ApplicationLiquid biopsy to identify mechanisms of drug resistance in cancer
• EGFR-mutant lung cancer is a subtype of non–small cell lung cancer (NSCLC) that exhibits sensitivity to EGFR TKIs such as erlotinib and gefitinib1
• However, acquired resistance develops after a median of 9–14 months1
• The most common mechanism of TKI resistance is a second-site mutation (T790M) in the EGFR kinase domain, which can be detected in >50% of biopsies done after resistance develops2
1Mok, T.S. et al. N. Engl. J. Med. 361, 947–957 (2009)
2Sequist, L.V. et al. Sci. Transl. Med. 3, 75ra26 (2011)
Landscape of resistance mutations in NSCLC
2Sequist, L.V. et al. Sci. Transl. Med. 3, 75ra26 (2011)
RS NGS part 3: Targeted NGS for Cancer Research 33
Sample to Insight
34
GeneRead Targeted Panels for NGS: ApplicationLiquid biopsy to Identify mechanisms of drug resistance in cancer
Power of liquid biopsy: Predict resistance before a solid biopsy is available
• AZD9291 is an oral, irreversible, mutant-selective EGFR TKI developed to have potency against tumors bearing EGFR activating mutations (for example, L858R or exon 19 deletion) in the presence of the T790M mutation
• Can we identify potential mechanisms of resistance to AZD9291 before the availability of resistance biopsy specimens?
• Use GeneRead targeted NGS panel to screen cell-free plasma DNA (cfDNA) for potential resistance mutations by comparing pretreatment and post-disease progression plasma specimens from a phase I clinical trialo cfDNA serves as a liquid biopsy to monitor disease progression by examining the landscape of
mutations before a resistance solid biopsy is available
May 2015
RS
Sample to Insight
GeneRead Targeted Panels for NGS: ApplicationLiquid biopsy to Identify mechanisms of drug resistance in cancer
Liquid biopsy approach
Collect 10-20 cc blood (pre-treatment (Baseline) and post progression)
Prepare plasma
Isolate cfDNA using the QIAmp Circulating Nucleic Acid Kit
Enrich targets (20 genes) using the GeneRead DNAseq Lung Cancer panel
Construct libraries
Ultra-deep sequencing (30,000x) on a HiSeq (2 × 100 bp)
RS NGS part 3: Targeted NGS for Cancer Research 35
Sample to Insight
GeneRead Targeted Panels for NGS: ApplicationLiquid biopsy to Identify mechanisms of drug resistance in cancer
Liquid biopsy: Proof of concept
Targeted NGS identified an acquired T→A mutation (green) in 1.3% of reads, encoding an EGFR C797S mutation.
RS NGS part 3: Targeted NGS for Cancer Research 36
Sample to Insight
GeneRead Targeted Panels for NGS: ApplicationLiquid biopsy to Identify mechanisms of drug resistance in cancer
Liquid biopsy: EGFR C797S mutation in progression/resistance to AZD9291 (TKI)
NGS of baseline and progressiontumor biopsies (top) confirmed the acquired C797S mutation detected with plasma NGS (bottom).
Plasma NGS detects the same T→A C797S mutation (green) found in the tumor and additionally detects a second G→C mutation encoding the C797S mutation (blue).
RS NGS part 3: Targeted NGS for Cancer Research 37
Sample to Insight
Thank you for attending today’s webinar!
Contact QIAGENCall: 1-800-426-8157
Email: [email protected]
Questions?
Thank You For Attending
NGS part 3: Targeted NGS for Cancer Research 38