Digital DNA-seq Technology: Targeted Enrichment for Cancer Research
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Transcript of Digital DNA-seq Technology: Targeted Enrichment for Cancer Research
Sample to Insight
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Mutational analysis using QIAGEN’s QIAseq® panels and Sample to Insight® NGS solutions
Raed Samara, PhDGlobal Product Manager, QIAGEN
QIAseq Targeted NGS for Cancer Research, 10.10.2016
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Sample to Insight
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Legal disclaimer
QIAseq Targeted NGS for Cancer Research, 10.10.2016
• 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.
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Precision medicine: Right drug, right patient, right time and dose
QIAseq Targeted NGS for Cancer Research, 10.10.2016
“One size fits all” does not work
Sample to Insight
Mutations
AGCTCGTTGCTCAGCTCReference genome
AGCTCGTTGCTCAGCGTTCInsertion
AGCTC---GCTCAGCTC
Deletion
Indels Copy number variations
T
G
CA
T GA
C
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DNA variants for precision medicine
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Sample to Insight
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Actionable DNA variants for precision medicine
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Mutations
AGCTCGTTGCTCAGCTCReference genome
AGCTCGTTGCTCAGCGTTCInsertion
AGCTC---GCTCAGCTC
Deletion
Indels Copy number variations
Only a handful of mutations are actionable
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
Sample to Insight
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Actionable DNA variants for precision medicine
QIAseq Targeted NGS for Cancer Research, 10.10.2016
How many?
Sample to Insight
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
KRAS25%
EGFR
23%
EML4-ALK6%
BRAF3%
PIKC3A3%
MET2%
ERBB2
1%
MAP2K10.4%
NRAS
0.2%
Un-know
n37%
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Precision medicine for lung cancer
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Current lung cancer biomarker landscape
• How many mutations to test for?
• How to test for these mutations
◦ Sequential testing
◦ Parallel testing
Adapted from: Govindan, R. et al. (2012). Genomic landscape of non-small cell lung cancer in smokers and never-smokers. Cell 150, 1121–34.
Sample to Insight
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
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Benefits of Targeted DNA Sequencing:
More relevant data
More cost effective
Detect low-frequency mutations
Lower DNA requirements
Higher multiplexing capabilities
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Actionable DNA variants for precision medicine
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Targeted DNA sequencing delivers accurate information required for precision medicine
Clinical utility requires targeted analysis
Sample to Insight
• Well-defined content
• Small target size
• More reads per sample
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Why choose targeted DNA sequencing?
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Targeted DNA sequencing limits the genes or targets to be sequenced
Features Benefits
• Examine variants that matter
• Multiplex many samples to save money
• Detect low frequency variants
Sample to Insight
Sample Insight
The principle of targeted enrichment is to simultaneously sequence millions of small DNA fragments that represent the region of interest
gDNA
Variants Report:
KRAS G12DEGFR T790MIDH1 R132H
KRASEGFRIDH1
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Sample isolation
Library construction & targeted enrichment
NGS run Data analysis Interpretation
Targeted DNA Sequencing (TDS)
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Shrink the genome
Sample to Insight
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Why choose PCR-based targeted enrichment?
QIAseq Targeted NGS for Cancer Research, 10.10.2016
• Offers specificity that beats capture-based approaches
Features Benefits
• Lets you use sequencing capacity on regions targeted by the panel, with minimal off-target sequencing
• Lets you achieve more uniform enrichment for more sequencing efficiency
It delivers unmatched specificity and uniformity (compared to capture-based methods)
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Sample Insight
• Turnaround time, and limited amounts of DNA
• Uniformity of enrichment
• Coverage of GC-rich regions
• Platform-dependent challenges
• Data processing & variant calling
• Isolation of high-quality DNA samples
• Quantification of amplifiable (not total) amounts of DNA
• Clinical & biological interpretation of data
RS
Sample QC Library QC
Variantconfirmation
Sample isolation
Library construction & targeted enrichment
NGS run Data analysis Interpretation
Sample to Insight: Integrated universal targeted NGS workflow
QIAseq Targeted NGS for Cancer Research, 10.10.2016
To overcome NGS challenges
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Inability to detect low-frequency mutations
Inefficient enrichment and sequencing of
GC-rich regions
PCR and sequencing errors• Limits sensitivity and accuracy of calling low-frequency variants
o Doesn’t let you confidently call variants down to 1% variant allele frequency (VAF)
Suboptimal uniformity of
enrichment and sequencing
Suboptimal, GC-rich region-incompatible PCR chemistry• Limits comprehensiveness of panel coverage
o Doesn’t let you efficiently sequence clinically-relevant genes such as CEBPA or CCND1 – or clinically-relevant regions such as TERT promoter
Conventional PCR protocols and two-primer amplicon design• Increases variability in coverage across targeted genomic regions
o Causes you to over-sequence to accommodate the under-sequencedo Doesn’t let you call variants in low-depth regions
Mainly due to inferior PCR amplification approaches
Challenges of current DNA targeted sequencing approaches
QIAseq Targeted NGS for Cancer Research, 10.10.2016
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DNA
dsDNA
PCR amplification & sequencing
PCR and sequencing errors
The necessary evil: PCR amplification
QIAseq Targeted NGS for Cancer Research, 10.10.2016
PCR amplification is required for target enrichment, but…
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5 reads OR library fragments that look exactly the same. Cannot tell whether they represent:
1. 5 unique DNA molecules, or
2. Quintuplets of the same DNA molecule (PCR duplicates)
Conventional targeted DNA sequencing
EGFR exon 21
Quantification based on non-unique reads does not reflect quantities of original DNA molecules
Challenges of conventional targeted DNA sequencing
QIAseq Targeted NGS for Cancer Research, 10.10.2016
PCR duplicates limit accurate quantification
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Conventional targeted DNA sequencing
EGFR exon 21
*
Variant calling based on non-unique reads does not reflect the mutational status of original DNA molecules
Challenges of conventional targeted DNA sequencing
QIAseq Targeted NGS for Cancer Research, 10.10.2016
A mutation is seen in 1 out of 5 reads that map to EGFR exon 21. Cannot accurately tell whether the mutation is:
1. A PCR or sequencing error (artifact) / false positives, or
2. A true low-frequency mutations
PCR and sequencing errors (artifacts) limit variant calling accuracy
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• Proprietary PCR chemistry to enrich even GC-rich regions
• Primers based on single primer extension (SPE) approach for enhanced uniformity
Panel box (kit)
QIAGEN’s solutions to overcome challenges of targeted NGS
QIAseq Targeted NGS for Cancer Research, 10.10.2016
QIAseq targeted DNA panels
• Molecularly-barcoded library adapters to incorporate unique molecular indices (UMIs).
Index box (kit)
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With the QIAseq targeted DNA panels, variant detection is done by analyzing unique DNA molecules instead of total reads
Overcoming current challenges
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Current approach ChallengesHow QIAseq targeted DNA panels overcome challenges of current approaches
• Conventional targeted DNA sequencing for variant detection
• PCR and sequencing errors • UMIs that enable digital sequencing to correct for PCR and sequencing errors
• Inefficient sequencing of GC-rich regions
• Proprietary chemistry to efficiently sequence GC-rich regions
• Suboptimal uniformity of enrichment and sequencing
• SPE-based primer design to increase uniformity
For optimal variant detection
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TATCGTACAGAT(12 nucleotides long)
Incorporate this random barcode (signature) into the original DNA molecules before amplification to preserve their uniqueness
What is a UMI (molecular barcode)?
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Tag (barcode) to identify unique DNA molecules
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DNA
dsDNA
TATCGTACAGAT
Molecularly-barcoded adapter Incorporate this random barcode (signature) into the original DNA molecules before amplification to preserve their uniqueness
PCR amplification & sequencing
Correct for PCR duplicates & errors
How are UMIs incorporated?
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Ligate molecularly-barcoded adapters to unique DNA molecules before amplification
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5 unique DNA molecules
since 5 molecular barcodes are detected
Quintuplets of the same DNA molecule (PCR duplicates)
since 1 molecular barcode is detected
UMI
Digital sequencing with UMIs
UMIs before any amplification
Achieve accurate quantification with molecular barcodes
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Count and analyze single original molecules (not total reads) = digital sequencing
5 reads OR library fragments that look exactly the same. Cannot tell whether they represent:
1. 5 unique DNA molecules, or
2. Quintuplets of the same DNA molecule (PCR duplicates)
Conventional targeted DNA sequencing
EGFR exon 21
Sample to Insight
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False variant is present in some fragments carrying the same UMI
True variant is present in all fragments carrying the same UMI
UMI
UMIs before any amplification
* *****
Digital sequencing with UMIs
Achieve accurate variant calling with molecular barcodes
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Conventional targeted DNA sequencing
EGFR exon 21
*A mutation is seen in 1 out of 5 reads that map to EGFR exon 21. Cannot accurately tell whether the mutation is:
1. A PCR or sequencing error (artifact) / false positives, or
2. A true low-frequency mutations
Count and analyze single original molecules (not total reads) = digital sequencing
Sample to Insight
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Sample isolation
Library construction & targeted enrichment
NGS run Data analysis InterpretationSample Insight
Panels and molecularly-
barcoded adapters
Barcode-aware variant calling
pipeline
QIAseq targeted DNA panels: Sample to Insight
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Panels, molecularly-barcoded adapters and data analysis algorithms
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Specifications of QIAseq targeted DNA panels
QIAseq Targeted NGS for Cancer Research, 10.10.2016
DNA input As little as 20 ng DNA
Primer multiplexing level 11,500 / 9600 primers (Catalog / Custom DNA)
Number of primer pools 1
Enrichment technology SPE-based with molecularly-barcoded adapters
Amplicon size Average 150 bp
Sample multiplexing level 384 (Illumina), 96 (Ion Torrent)
Total workflow time 8–9 hours
Number of libraries per sample 1
Sequencer compatibility Illumina and Ion Torrent platforms
Variant allele frequency called 1%
Sample to Insight
End repair and A tailing
Adapter ligation / library construction (incorporation of adapters, molecular barcodes and sample indexes)
5’5’
5’
5’
MB
MB
Adapter
5’
IL-FRSP
Add GSPs and UP*
5’IL-U SIP
Add indexes and UP*
Universal PCR amplificationSample indexing and amplification
Sequencing-ready library
MB: Molecular barcodeRSP: Region-specific primerFP: Forward primerUP: Universal primerSIP: Sample index primer
AA
5’MB
Target enrichment by SPE5’
*Preceded by bead cleanup
Lib quant*
Enzyme-based random DNA fragmentation
DNA
5’5’
1.5
Day
s
QIAseq Targeted DNA Panel: Workflow (Illumina®)
QIAseq Targeted NGS for Cancer Research, 10.10.2016 25
Sample to Insight
QIAseq Targeted DNA Panel: Workflow (Ion Torrent™)
QIAseq Targeted NGS for Cancer Research, 10.10.2016 26
End repair and A tailing
Adapter ligation / library construction (incorporation of adapters, molecular barcodes and sample indexes)
5’5’
5’
5’
MB
MB
Adapter
5’
LT-FRSP
Add GSPs and UP*
5’LT-U P1
Add indexes and UP*
Universal PCR amplificationSample indexing and amplification
Sequencing-ready library
MB: Molecular barcodeRSP: Region-specific primerFP: Forward primerUP: Universal primerP1: P1 primer
AA
5’MB
Target enrichment by SPE5’
*Preceded by bead cleanup
Lib quant*
Enzyme-based random DNA fragmentation
DNA
5’5’
1.5
Day
s
Sample to Insight
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1. Exonic regions of genes plus 10 bases to cover intron / exon junctions
2. Mix of type of coverage 1 (for tumor suppressor genes) and HotSpots for oncogenes
3. SNPs
4. Full chromosome
QIAseq targeted DNA panels
QIAseq Targeted NGS for Cancer Research, 10.10.2016
PanelVariant (Cat) number
Number of genes
Number of primers
Type of coverage
Breast cancer panel DHS-001Z 93 4831 1
Colorectal cancer panel DHS-002Z 71 2929 1
Myeloid Neoplasms panel DHS-003Z 141 5887 1
Lung cancer panel DHS-005Z 72 4149 1
Actionable solid tumor panel DHS-101Z 23 651 2
BRCA1 and BRCA2 panel DHS-102Z 2 223 1
BRCA1 and BRCA2 Plus panel DHS-103Z 6 348 1
Pharmacogenomics panel DHS-104Z 39 146 3
Mitochondria panel DHS-105Z Chromosome M 222 4
Inherited diseases panel DHS-3011Z 298 11,579 1
Comprehensive cancer panel DHS-3501Z 275 11,311 1
List of panels
Types of coverage:
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QIAseq targeted DNA panels
QIAseq Targeted NGS for Cancer Research, 10.10.2016
List of panels
PanelVariant (Cat) number
Panel size (bases)
Specificity (reads with primers, %)
Uniformity (0.2x mean baseMT, %)
Breast cancer panel DHS-001Z 370,942 96.47 99.84
Colorectal cancer panel DHS-002Z 215,328 90.39 99.79
Myeloid Neoplasms panel DHS-003Z 436,672 95.31 99.71
Lung cancer panel DHS-005Z 318,059 97.3 99.91
Actionable solid tumor panel DHS-101Z 15,160 90.48 99.85
BRCA1 and BRCA2 panel DHS-102Z 16,405 99.59 100
BRCA1 and BRCA2 Plus panel DHS-103Z 25,590 99.46 99.92
Pharmacogenomics panel DHS-104Z 3313 93.43 99.34
Mitochondria panel DHS-105Z 16,570 99.72 99.08
Inherited diseases panel DHS-3011Z 838,627 97.29 99.21
Comprehensive cancer panel DHS-3501Z 836,670 97.42 199.76
Uniformity and specificity are defined based on NA12878 tests
Sample to Insight
Extended and Custom
What is the list of your targets?
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Extended panels
Custom panels
• Extend the contents of an existing cataloged panel• Turnaround time = 14 days
• Bioinformatically target any gene(s) or genomic region(s) within the human genome
• Turnaround time = 14 days
Customized panels
QIAseq Targeted NGS for Cancer Research, 10.10.2016
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CEBPA
GC content
Coverage
GC content
Coverage
CCND1
The proprietary PCR chemistry used in the QIAseq targeted DNA panels enables efficient coverage of regions high in GC content
Comprehensive coverage of GC-rich regions
QIAseq Targeted NGS for Cancer Research, 10.10.2016
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830 kb region was enriched from 20 ng of NA12878 DNA with Comprehensive Cancer Panel. Library was constructed for sequencing on a MiSeq, with 2600x read depth. The panel achieved a uniformity of 99.5% at 0.2x of mean coverage, and 98% at 0.3x of mean coverage.
Unmatched uniformity
QIAseq Targeted NGS for Cancer Research, 10.10.2016
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Benefits of QIAseq targeted DNA panels
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Feature Benefit
Low DNA input (as low as 20 ng DNA) Preserve sample
High primer multiplexing capability (up to 10,000 primers) Detect a large number of DNA variants
Single pool of primers Easier sample handling
SPE-based target enrichment Flexibility in primer design
Small amplicons (average size 150 bp) Generate relatively small library fragments to maintain compatibility with fragmented DNA (FFPE and ctDNA samples)
High sample multiplexing (up to 384 samples) Increased sample throughput to decrease sequencing costs
Automation-friendly workflow Streamline operations for high throughput
Molecular barcode-aware variant caller Confidently call low-frequency mutations
Suite of complementary data analysis tools Save resources
Affordable per-sample cost Save $$$
All required reagents (including beads) in 2 kits Simplified logistics & ordering
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This online GenomeWeb seminar focused on the design of a large cohort study for assessing breast cancer risk and how using an innovative digital sequencing approach is able to solve the previously unmet challenges of this type of NGS study design.
Fergus J. Couch, PhD
Professor and Chair Division of Experimental Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic
https://genomeweb.webex.com/genomeweb/lsr.php?RCID=30c8e3fe698b7feca1ed79ac117fbed0
Application: Large cohort study for breast cancer risk
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Sequencing 60,000 samples
Sample to Insight
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Sample to Insight: Integrated universal targeted NGS workflow
QIAseq Targeted NGS for Cancer Research, 10.10.2016
To overcome NGS challenges
Sample Insight
• Turnaround time, and limited amounts of DNA
• Uniformity of enrichment
• Coverage of GC-rich regions
• Platform-dependent challenges
• Data processing & variant calling
• Isolation of high-quality DNA samples
• Quantification of amplifiable (not total) amounts of DNA
• Clinical & biological interpretation of data
Sample QC Library QC
Variantconfirmation
Sample isolation
Library construction & targeted enrichment
NGS run Data analysis Interpretation
Sample to Insight
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Barcode-aware variant caller has been developed
Caller is available on the cloud
In conjunction with molecular barcodes incorporated in the workflow, the caller can confidently call low-frequency variants (down to 1% variant allele frequency, “VAF”)
Variant caller will do the following:• Mapping
• Alignment
• Molecular barcode counting
• Variant / calling
• Variant / annotation – variants based on public databases
Data analysis with barcode-aware variant caller – overview
QIAseq Targeted NGS for Cancer Research, 10.10.2016
For QIAseq targeted DNA panels
Sample to Insight
Data analysis with barcode-aware variant caller – overview
FASTQ or BAM files are uploaded into cloud-based data analysis portal
The following inputs are needed (by customer):• Set up file
• Panel used
• File laneso 1-lane (MiSeq/HiSeq/NextSeq concatenated)o 4-lane (NextSeq individual lane files)
Inputs
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Data analysis with barcode-aware variant caller – overview
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Outputs
Summary file• Stats
o Specificityo Uniformityo Molecular barcode counts
• Variantso Frequencyo Annotations
VCF
Sample to Insight
Actionable solid tumor
Disease-specific Comprehensive
DetectionDiscovery
MultiplexingTarget size
Custom & extended
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 discovery
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Why choose targeted DNA sequencing?
QIAseq Targeted NGS for Cancer Research, 10.10.2016
Detect known variants & discover novel variants
Sample to Insight
Thank you for attending today’s webinar!Contact QIAGENCall: 1-800-426-8157
Email: [email protected]
Questions?
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Thank you for attending
QIAseq Targeted NGS for Cancer Research, 10.10.2016