Molecular analysis via next-generation sequencing - National
Transcript of Molecular analysis via next-generation sequencing - National
Sample & Assay Technologies
Molecular analysis via next-generation sequencing
NMG Annual Fall Meeting, Toronto, Canada
Raed Samara, PhD, PMP
Global Product Manager
October 21, 2013
Sample & Assay Technologies Legal Disclaimer
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• 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 & Assay Technologies Agenda
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1. Primer on NGS
2. QIAGEN’s Preanalytic solutions to NGS challenges: Sample-to-Insight
3. Application example (Sequencing of FFPE cancer samples)
4. Considerations for using NGS in Molecular Microbiology
Sample & Assay Technologies Agenda
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1. Primer on NGS
2. QIAGEN’s Preanalytic solutions to NGS challenges: Sample-to-Insight
3. Application example (Sequencing of FFPE cancer samples)
4. Considerations for using NGS in Molecular Microbiology
Sample & Assay Technologies What is Next-Generation Sequencing (NGS)?
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Massively Parallel Sequencing
Instead of sequencing a DNA sequence from
Sequence many small pieces at the same time
Resulting in the original sequence
(with overlaps removed)
.Target DNA is fragmented
Adaptors ligated to fragments
(Library construction)
Clonal amplification of fragments
.Direct step-by-step detection of
each nucleotide base
incorporated during the
sequencing reaction
Starting gDNA
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Sample & Assay Technologies NGS needs in Molecular Analysis
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Simple, but integrated workflow
Work with low quality and qualntity DNA samples
Focus efforts on a focused set of genes important to molecular analysis (save
time) + Selective sequencing saves sequencing capacity (save $$$)
Identify low frequency DNA variations
Simple methodology to make variant calls
Sample Insight
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Sample & Assay Technologies Agenda
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1. Primer on NGS
2. QIAGEN’s Preanalytic solutions to NGS challenges: Sample-to-Insight
3. Application example (Sequencing of FFPE cancer samples)
4. Considerations for using NGS in Molecular Microbiology
Sample & Assay Technologies QIAGEN’s solutions to NGS workflow challenges
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Sample Insight
Raed Samara, PhD; NMG Conference; 10/21/2013
Sample & Assay Technologies QIAGEN’s solutions to NGS workflow challenges
Three major components in workflow
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Platform Pre-Analytics
Sample Insight
Software
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Sample & Assay Technologies QIAGEN’s solutions to NGS workflow challenges
….. but several steps in each component of the workflow
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Platform
Sample
Isolation
Target Enrichment
Library Preparation
Library Quantification
NGS
Run
Data
Analysis
Pre-Analytics
Sample Insight
Biological Insight
Software
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Sample & Assay Technologies QIAGEN’s solutions to NGS workflow challenges
Each step has its own challenges
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Platform
Sample
Isolation
Target Enrichment
Library Preparation
Library Quantification
NGS
Run
Data
Analysis
Pre-Analytics
Sample Insight
Biological Insight
Software
DNA Quality Library Quality Platform-specific Data processing Variant calling
Biological Interpretation of
data
Efficiency & Yield
Raed Samara, PhD; NMG Conference; 10/21/2013
Content AND Coverage,
Specificity & Uniformity
Sample & Assay Technologies QIAGEN’s solutions to NGS workflow challenges
Major challenge: Fragmented workflow
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Platform
Sample
Isolation
Target Enrichment
Library Preparation
Library Quantification
NGS
Run
Data
Analysis
Pre-Analytics
Sample Insight
Biological Insight
Software
DNA Quality Library Quality Platform-specific Data processing Variant calling
Biological Interpretation of
data
Dedicated products in a complete workflow to overcome challenges of your
Preanalytic DNA NGS workflow
Efficiency & Yield
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Content AND Coverage,
Specificity & Uniformity
Sample & Assay Technologies QIAGEN’s solutions to NGS workflow challenges
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Platform
Sample
Isolation
Target Enrichment
Library Preparation
Library Quantification
NGS
Run
Data
Analysis
Pre-Analytics
Sample Insight
Biological Insight
Software
DNA Quality Library Quality Platform-specific Data processing Variant calling
Biological Interpretation of
data
Efficiency & Yield
Raed Samara, PhD; NMG Conference; 10/21/2013
Content AND Coverage,
Specificity & Uniformity
Sample & Assay Technologies QIAGEN — the gold-standard in DNA purification
The best DNA quality possible for outstanding NGS results
High-quality purification from virtually all sample types
Sample type Kit
FFPE tissue QIAamp DNA FFPE Kit
Blood QIAamp DNA Blood Mini Kit
Tissues, swabs, CSF, body fluids QIAamp DNA Mini Kit
High-molecular weight DNA MagAttract HMW DNA Kit
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Automated on QIAcube:
Consistency and standardization
Sample & Assay Technologies QIAGEN’s solutions to NGS workflow challenges
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Platform
Sample
Isolation
Target Enrichment
Library Preparation
Library Quantification
NGS
Run
Data
Analysis
Pre-Analytics
Sample Insight
Biological Insight
Software
DNA Quality Library Quality Platform-specific Data processing Variant calling
Biological Interpretation of
data
Efficiency & Yield
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Content AND Coverage,
Specificity & Uniformity
Sample & Assay Technologies Three levels of DNA Sequencing
Targeted Exome
Sequencing
(Genes Panels)
Whole Exome
Sequencing
Whole Genome
Sequencing Cost
Data Complexity
Irrelevant data
Starting DNA material
Poor quality reads
Deep sequencing
Multiplexing
Quality of reads
Coverage
Gene Panel Sequencing: Efficient, Effective and Economical method
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Sample & Assay Technologies Traditional NGS Workflow
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Sample & Assay Technologies New NGS Workflow with Targeted Enrichment
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• Focused on your genes of interest
• Why look at all 20,000
genes in the human
genome when you are
interested in only a few?
• More confidence and deeper
coverage
• Enables deep sequencing to ID
low frequency / rare variants
• Integrated controls to assess
target enrichment
Achieve more sensitive variant
detection with 1 additional step
80 ng
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Sample & Assay Technologies Shrink the Genome
Focus on Genes of interest relevant to disease
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Sequencing Information Cost per
sample
Coverage No. of
samples
multiplexed
DNA input
Genome 3 x 109 bps - 30X N/A 1µg
Exome 5X 107 bps $2000 100X 1 500 ng- 1µg
30 Genes 6 x 104 bps $200 1000X 12 80 ng
* 7.5 GB sequencing capacity
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Sample & Assay Technologies Targeted Exome Enrichment: Principles
Multiplex PCR-enabled enrichment of gene of interest
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Exon 1 Exon 2 Exon 1 Exon 2 Exon 1 Exon 2
gDNA
GOI 3 GOI 2 GOI 1
BRAF Akt1 ErbB2
Each exon of
GOI is targeted
by several
overlapping
primers
Targeted Exome Enrichment allows you to focus on you GOI
The entire collection
of primers is divided
into 4 tubes, each
containing non-
adjacent primer sets
Contains a set
of endogenous
controls (for use
in QC)
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Sample & Assay Technologies Targeted Exome Enrichment: Principles
Simple Protocol – 2 hours
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GOI 3 GOI 2 GOI 1
BRAF Akt1 ErbB2
Exon 1 Exon 2 Exon 1 Exon 2 Exon 1 Exon 2
Isolated
gDNA
GeneRead DNASeq
Master Mix
GeneRead DNASeq
Panels
Total of 4
PCR
reactions
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Template for
Clonal
Amplification
Pooled
Amplicons Prepare for sequencing
(Library Preparation)
Wet-bench verification – we confirm that primers work
All of the amplicons of your GOI in ONE tube
Sample & Assay Technologies Performance metrics
High coverage, specificity, and uniformity
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Panel 1 (6 genes): Custom GeneRead
DNAseq Gene Panel
Panel 2 (20 genes): Human Lung Cancer
GeneRead DNAseq Gene Panel
Panel 3 (124 genes): Human Comprehensive
Cancer GeneRead DNAseq Gene Panel
Specific
ity (
% o
n-t
arg
et re
ads)
Design coverage (≥90%) Specificity (on-target reads; ≥85%)
Coverage uniformity
(≥85% with 0.1X of median sequencing depth)
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Panel 1 (6 genes) Panel 2 (20 genes)
Panel 3 (124 genes)
% C
overe
d b
y d
esig
n
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Panel 1 (6 genes) Panel 2 (20 genes)
Panel 3 (124 genes)
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Panel 1 (6 genes) Panel 2 (20 genes)
Panel 3 (124 genes)
Covera
ge u
niform
ity
Sample & Assay Technologies Biologically/Clinically relevant gene panels
GeneRead DNAseq Gene Panels
Comprehensive Cancer Panel
Disease Focused Gene Panels
Breast cancer
Colon Cancer
Gastric cancer
Leukemia
Onco-hematology (coming soon)
Cardiomyopathy (coming soon)
Inheritance disease Panel (coming soon)
Actionable Mutation Cancer Panel (coming soon)
Clinically-relevant Mutation Tumor Panel (coming soon)
Cancer Predisposition Panel (coming soon)
Custom Content
ANY GENE or COLLECTION OF
GENES in Human Genome
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Liver cancer
Lung Cancer
Ovarian Cancer
Prostate Cancer Cancer Genome Census
ClinVar
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Sample & Assay Technologies
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GeneRead DNAseq Custom Panel
Custom Content
ANY GENE or COLLECTION OF
GENES in Human Genome
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Sample & Assay Technologies Breast Cancer Gene Panel
Description
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Sample & Assay Technologies Breast Cancer Gene Panel
Somatic: All
Germline and Somatic: APC, BRCA1, BRAC2, TP53
With approved FDA inhibitor: BRAF, EGFR, ERBB2
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Sample & Assay Technologies Advantages of Targeted Exon Sequencing
Lower Cost:
– Cost of whole genome sequencing is still high
Biology:
– Disease associated genetic varaiants are limited to a
small number of genes
Results:
– Enable deep sequencing, ideal to identify or discover low
frequency variants
Clinical Clinical research
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Sample & Assay Technologies QIAGEN’s solutions to NGS workflow challenges
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Platform
Sample
Isolation
Target Enrichment
Library Preparation
Library Quantification
NGS
Run
Data
Analysis
Pre-Analytics
Sample Insight
Biological Insight
Software
DNA Quality Efficiency &
Yield Library Quality Platform-specific
Data processing Variant calling
Biological Interpretation of
data
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Content AND Coverage,
Specificity & Uniformity
Sample & Assay Technologies Library Preparation
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End Repair Adapter ligation
Size selection & Clean up
Sample
Isolation
Target Enrichment
Library Preparation
Library Quantification
NGS
Run
Data
Analysis
Biological Insight
Pooled
amplicons
Sequencing-
ready
amplicons
Sample & Assay Technologies
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GeneRead DNA Library Prep Kits
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Automated on QIAcube:
High reproducibility
No handling variability
Fast procedure that allows
up to 50% time savings
Faster, less variable & more efficient workflow
Sample & Assay Technologies GeneRead DNA Library Prep Kits
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High yield of Library DNA
Uniform coverage distribution
Input DNA as little as 50 ng
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Sample & Assay Technologies Automated Spin column-based Size Selection
Before size selection
Adapter dimers
Adapter monomers
Proven silica-technology delivers precise size selection
Efficient removal of small DNA fragments and adapter-dimers
No tedious bead handling or risk of ethanol carry-over
Can be automated on the QIAcube
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Clean up your library before sequencing
Spend your sequencing capacity on amplicons not adapters
GeneRead size selection
Sample & Assay Technologies QIAGEN’s solutions to NGS workflow challenges
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Platform
Sample
Isolation
Target Enrichment
Library Preparation
Library Quantification
NGS
Run
Data
Analysis
Pre-Analytics
Sample Insight
Biological Insight
Software
DNA Quality Library Quality Platform-specific Data processing Variant calling
Biological Interpretation of
data
Efficiency & Yield
Raed Samara, PhD; NMG Conference; 10/21/2013
Content AND Coverage,
Specificity & Uniformity
Sample & Assay Technologies Library Quantification & Qualification
Why quantify & qualify your library?
Too much template
Mixed signals and un-resolvable data
Too little template
Reduced sequencing coverage
Reduced read depth
Empty runs
Increase cost per run
Wasted time
Save money and time by running libraries of adequate
quality ONLY
Simple out-of-the-box solution to:
Quantify to ensure uniform starting concentrations of libraries
Qualify to ensure high quality reads
GeneRead DNAseq
Library Quantification
Array
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Sample & Assay Technologies GeneRead DNAseq Library Quantification Array
Workflow: Quantification and QC in a single PCR run
QC score QC result Proceed?
1–4 Pass Yes
4–8 Marginal With Caution
>8 Fail No
Enables consistent results with every NGS run
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Sample & Assay Technologies GeneRead DNASeq Library Quantification Array
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An Example
Segregate bad libraries before sequencing; Save money and time
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Sample & Assay Technologies QIAGEN’s solutions to NGS workflow challenges
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Platform
Sample
Isolation
Target Enrichment
Library Preparation
Library Quantification
NGS
Run
Data
Analysis
Pre-Analytics
Sample Insight
Biological Insight
Software
DNA Quality Library Quality Platform-specific Data processing Variant calling
Biological Interpretation of
data
Efficiency & Yield
Raed Samara, PhD; NMG Conference; 10/21/2013
Content AND Coverage,
Specificity & Uniformity
Sample & Assay Technologies DNAseq Panels compatible with NGS platforms
No manipulation of target enrichment protocols needed
Ion Illumina
• MiSeq
• GA Iix
• HiSeq 1000
• HiSeq 2000
• HiSeq 1500
• HiSeq 100
• Torrent PGM
• 314
• 316
• 318
• Proton
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Sample & Assay Technologies QIAGEN’s solutions to NGS workflow challenges
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Platform
Sample
Isolation
Target Enrichment
Library Preparation
Library Quantification
NGS
Run
Data
Analysis
Pre-Analytics
Sample Insight
Biological Insight
Software
Content AND Coverage,
Specificity & Uniformity
DNA Quality Library Quality Platform-specific Data processing Variant calling
Biological Interpretation of
data
Efficiency & Yield
Raed Samara, PhD; NMG Conference; 10/21/2013
Sample & Assay Technologies
Data Analysis: Free, Complete and Easy to Use
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Primary level
Secondary level
Sample & Assay Technologies Results in Multiple Analysis Format
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Run Summary Specificity
Coverage
Uniformity
Numbers of SNPs and Indels
Summary By Gene Specificity
Coverage
Uniformity
# of SNPs and Indels
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Primary data analysis
Sample & Assay Technologies
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Features of Variant Report
dbSNP and
COSMIC ID
Predicted amino
acid change
Effect of SNP Impact of SNP
Link to qPCR
somatic mutation
assay
SNP detection
Indel detection
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Secondary data analysis
Sample & Assay Technologies QIAGEN’s solutions to NGS workflow challenges
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Platform
Sample
Isolation
Target Enrichment
Library Preparation
Library Quantification
NGS
Run
Data
Analysis
Pre-Analytics
Sample Insight
Biological Insight
Software
DNA Quality Library Quality Platform-specific Data processing Variant calling
Biological Interpretation of
data
Efficiency & Yield
Raed Samara, PhD; NMG Conference; 10/21/2013
Content AND Coverage,
Specificity & Uniformity
Sample & Assay Technologies Fast biological Interpretation with up-to-date scientific knowledge
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Tertiary data analysis
Sample & Assay Technologies Verify your NGS results
Complete Systems downstream of NGS
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Product
Line
Technology
Detection of
RT2 Profiler
SYBR Green qPCR
mRNA
qBiomarker
ARMS qPCR
Somatic Mutations
PyroMark
Pyrosequencing
Mutations, Variants, and Methylation
Complete workflow: Sample-to-Insight solutions
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Microbial
ARMS qPCR
Microbes
Sample & Assay Technologies Microbial DNA qPCR Multi-Assay Kits
28 focused kits around the pathogenicity of a specific microbe
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Bartonella henselae Pathogenicity
Bordetella pertussis Pathogenicity
Brucella spp Pathogenicity
Campylobacter jejuni Pathogenicity
Chlamydia trachomatis Pathogenicity
Clostridium difficile Pathogenicity
Clostridium perfringens Pathogenicity
C. diphtheriae Pathogenicity
Enterococcus faecalis Pathogenicity
EHEC Pathogenicity
Haemophilus influenzae Pathogenicity
Helicobacter pylori Pathogenicity
Legionella pneumophila Pathogenicity
Listeria monocytogenes Pathogenicity
Mycobacterium spp Pathogenicity
Neisseria meningitidis Pathogenicity
Pseudomonas aeruginosa Pathogenicity
Salmonella enterica Pathogenicity
Shigella dysenteriae Pathogenicity
Strepto. agalactiae Pathogenicity
Strepto. pneumoniae Pathogenicity
Streptococcus pyogenes Pathogenicity
Vibrio cholerae Pathogenicity
Yersinia enterocolitica Pathogenicity
Yersinia spp Pathogenicity
Laboratory Rodent Testing
MRSA: Methicillin-resistant S. aureus
SHV Antibiotic Resistance Genes
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Sample & Assay Technologies Microbial DNA qPCR Arrays
Profile or Identify the presence of microbial DNA with any array
Identification experiment answers the following question:
Are any of these microbes or genes present in the sample?
Must be compared against a known negative sample.
Answers are Yes or No.
Profiling experiment answers the following question:
Have the amounts of any of these microbes or genes changed?
Must be compared against a reference sample.
Answers are fold change.
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Sample & Assay Technologies Microbial DNA qPCR Array content
14 Arrays
Antibiotic Resistance Genes
Bacterial Vaginosis
Biodefense
Food testing: Dairy
Food testing: Meat
Food testing: Poultry
Food testing: Seafood
Food testing: Vegetable
Intestinal infections
Respiratory Infections
Sepsis
Urinary Tract Infections
Vaginal Flora
Water Analysis
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Sample & Assay Technologies Agenda
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1. Primer on NGS
2. QIAGEN’s Preanalytic solutions to NGS challenges: Sample-to-Insight
3. Application example (Sequencing of FFPE cancer samples)
4. Considerations for using NGS in Molecular Microbiology
Sample & Assay Technologies Genetic variant analysis in FFPE lung adenocarcinoma samples
Detection of Mutations in FFPE Lung Adenocarcinoma Samples
gDNA isolated from 3 FFPE lung adenocarcinoma and one FFPE normal lung samples
GeneRead Lung Cancer Gene Panel was used to enrich 20 genes
Library preparation, quantification and sequencing
QIAGEN NGS Data Analysis Web Portal
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Question: What are the mutations present in 3 similar Lung Cancers?
Experimental Design
Sample & Assay Technologies
Run Summary
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Sample Tumor
Sample-1
Tumor
Sample-1
Tumor
Sample-1
Normal
Sample
Total number of reads 1,629,983 1,892,621 1,851,514 1,625,119
Specificity
(Reads on target) 92% 92% 90% 90%
Median Coverage 608 744 703 577
Uniformity
(regions with >10% of
median coverage)
93% 92% 90% 93%
No. of SNPs 84 72 87 83
No. of Indels 8 11 9 6
Genetic variant analysis in FFPE lung adenocarcinoma samples
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Sample & Assay Technologies
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Detection of low frequency variant in lung adenocarcinoma
Genetic variant analysis in FFPE lung adenocarcinoma samples
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• Low-quality DNA samples (FFPE): ~1% DNA good enough quality for analysis
• Focus efforts on a focused genes: Save time and money
• 20 Gene Panel on 20 Samples = 1.6 GB of data (WG analysis = 150-200 GB)
Sample & Assay Technologies
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Data validation: PCR Assay
0
1
2
3
4
5
6
Tumor Sample-1
Tumar sample-2
Tumor Sample-3
Normal Sample
Ct
valu
e
Validation of EGFR:C499Y mutation by qBiomarker Somatic Mutation Assay
EGFR c.1496G>A (p.C499Y)
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Sample & Assay Technologies
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Tumor sample-1
(KRAS c.35G>T 35%)
Mutation analysis of codons 12 of KRAS using PyroMark Q24. Upper Pyrogram show G to T mutation in position 1 of
codon 12 of KRAS in lung adenocarcinoma sample. The mutation rate (35%) is similar to the NGS results (38%) confirming
the reliability of GeneRead DNAseq Gene Panel. The lower Pyrogram shows normal genotype in normal sample-1.
Normal sample-1
(KRAS c.35G>T 0%)
Data Validation: PyroMark Assay
Validation of KRAS:G12V somatic mutation by pyrosequencing assay
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Sample & Assay Technologies Agenda
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1. Primer on NGS
2. QIAGEN’s Preanalytic solutions to NGS challenges: Sample-to-Insight
3. Application example (Sequencing of FFPE cancer samples)
4. Considerations for using NGS in Molecular Microbiology
Sample & Assay Technologies Considerations for using NGS in Molecular Microbiology
How will you isolate DNA?
How will you amplify and purify DNA?
Will you be targeting the 16S rRNA or a different gene?
How big is the genome?
How much coverage do you need?
How much data do you need?
Which chemistry/instrument to use?
Which reference genome to use?
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Sample & Assay Technologies Summary
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Sample
Isolation
Target Enrichment
Library Preparation
Library Quantification
NGS
Run
Data
Analysis
Sample Insight
Biological Insight
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• Isolate high quality DNA to ensure successful sequencing runs
• Enrich for your GOI to enable deeper and more confident sequencing
• Achieve higher library yields to generate higher coverage
• Quantify libraries to achieve consistent results
• Qualify libraries to save money
• Identify variants easily to save time
• Use a comprehensive knowledge database to gain biological insight