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Sample & Assay Technologies
GeneRead DNAseq Targeted Exon Enrichment & GeneRead Library Quantification System for
Next Generation Sequencing
Shankar Sellappan, Ph.D.Global Product Manager
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Sample & Assay Technologies Pathway-Focused Analysis Tools
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Sample & Assay Technologies GeneRead DNAseq & Library Quant NGS System
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Agenda
� Introduction� Targeted Enrichment
� Workflow� Principles� Data Analysis� Pathway Content� Performance Data� Application Example
� Library Quantification� Workflow� Application Example
� Summary
Sample & Assay Technologies Biological Markers Define Biological Processes
Cell cycle Angiogenesis
P53MDM2CyclinsCDKs
VEGFbFGF
ANGPTPDGF
IL8TLRIFNγTNFβ
Ind
ivid
ual
p
arti
cip
ants
Inflammation
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Sample & Assay Technologies Complete Biological Story
Built on Pathway / Network Analysis
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Pat
hw
ay
Cell cycle Angiogenesis Inflammation
Sample & Assay Technologies Determining DNA Differences
Patient sample = ATGCCATCTGGGACGGGTCAGTAGWild-type = ATGCCATCTGTGACGGGTCAGTAG
How could that SINGLE base difference make the person sick?
Let’s look at the amino acids that are translated in both samples:
Patient sample DNA = ATG CCA TCT GGG ACG GGT CAG TAGAmino Acid Sequence = Met P S G T G Q Stop
Compare the two amino acid sequences:
Patient sample AA Sequence = Met P S G T G Q StopWild-Type AA Sequence = Met P S V T G Q Stop
Valine Glycine
If the wild-type protein, with the Valine positioned here, looked like:
and in the patient sample, it is a Glycine, and it looks like:
Well….the protein just won’t work.
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Sample & Assay Technologies What is NGS (Next Generation Sequencing)?
Massively Paralleled Sequencing
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Instead of sequencing a DNA sequence from
Sequence many small pieces at the same time
Sample & Assay Technologies When doing NGS analysis, what are you looking for?
Easy-to-use workflow and Data output
Detection of Low Prevalence Somatic Mutation in FFPE Lung Adenocarcinoma Sample
Human Lung Cancer GeneRead DNASeq Gene panel was used to enrich 20 genes in genomic DNA isolated from three FFPE lung adenocarcinoma and one FFPE normal lung samples. Sequencing data was analyzed using QIAGEN NGS Data Analysis Web Portal and high quality variants were filtered.
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Sample & Assay Technologies Your NGS research needs
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� Identify low frequency DNA mutation variants�Work with low quality DNA samples, such as FFPE
samples�Focus efforts on a focused set of genes important to
their research �Simple methodology to make variant calls�Selective sequencing saves sequencing capacity
Sample & Assay Technologies Traditional NGS Workflow
Isolate DNA
Library Prep & Quantification
NGS
Sequence Analysis & Variant ID
• Whole genome analysis• Too much irrelevant data• Poor quality reads / coverage
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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?
• Enables deep sequencing to ID low frequency mutation / rare variants
• Integrated controls to assess target enrichment
Achieve more sensitive mutation detection with 1 additional step
80 ng
Sample & Assay Technologies Target Enrichment: Principles
Multiplex PCR-enabled enrichment of gene of interest
Division of non-adjacent gene primer sets into 4 tubes increases amplification specificity.
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We provide primer sets that produce overlapping PCR products• For any gene or set of genes in the human genome
Sample & Assay Technologies Targeted Enrichment: Details
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~ 150 bp PCR products
Sample & Assay Technologies Complete Analysis Workflow
With integrated controls to assess sample quality / TE process
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- QIAGEN GeneRead Library Preparation Kits (for IT and IL)- QIAGEN GeneRead Size Selection Kits- QIAGEN GeneRead Library Indexing Kits
Coming Soon!
- QIAGEN NGS Platform
Sample & Assay Technologies NGS Sequence Analysis and Variant ID Software
� FREE Complete & Easy to use Data Analysis with Web-based Software
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Sample & Assay Technologies Read Mode: Paired End vs Single End
Goal: Increased # of Reads / Amplicon
TACGCATCGATGCGGTAACTGCTGATCGTCGTAGTGCTAGCTGA
Single End Sequencing:
Paired End Sequencing (both ends are sequenced):
Sequence of Interest:
TACGCATCGATGCGGTAACTGCTGATCGTCGTAGTGCTAGCTGA
TACGCATCGATGCGGTAACTGCTGATCGTCGTAGTGCTAGCTGA
AGTCGATCGTGATGCTGCTAGTCGTCAATGGCGTAGCTACGCAT
Sample & Assay Technologies How Genes on Panels Are Selected
Genes Involved in Disease
Genes with High Relevance
� Comprehensive Cancer Panel (124 genes)
� Disease Focused Gene Panels (20 genes)
� Breast cancer
� Colon Cancer
� Gastric cancer
� Leukemia
� ALSO AVAILABLE: Custom Panels from ANY GENE or COLLECTION OF GENES in Human Genome
� Biologically relevant gene content� Clinically relevant: Published association with the
disease state – Multiple Publically accessible databases– Text mining tools– Manually curated
� Technically relevant gene content� Most frequently mutated genes� Specific feedback from the thought leaders
Liver cancer
Lung Cancer
Ovarian Cancer
Prostate Cancer
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Sample & Assay Technologies Targeted Enrichment: Panel Information
Sample & Assay Technologies NGS Target Enrichment Design Strategy
Commonly encountered issues solved by GeneRead Algorithm
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• Design Coverage• How much of the gene is covered by your amplicons
• Sequence Coverage Uniformity• How much ease base pair is covered: Ideally, it should be uniform
• Specificity• % of reads mapped back to your sequence of interest
Sample & Assay Technologies Design Coverage Information Provided
Base Pairs Covered
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Sample & Assay Technologies Coordinate Information
Sample & Assay Technologies BED File Information
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C1 = Covered Regions
C*1 = Uncovered Regions
Sample & Assay Technologies Gene Visualization with UCSC Genome Browser
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Instructions will be provided online at SABiosciences.com
Sample & Assay Technologies GeneRead DNAseq Gene Panel: Performance data
Design coverage
% C
ove
red
by
Des
ign
Discover more potential variants by covering more exons for genes of interest in assay design
GeneRead DNAseq Custom Panel
GeneRead DNAseq Lung Cancer Panel
GeneRead DNAseq Comprehensive Cancer Panel
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Sample & Assay Technologies
Sequence coverage uniformity
More bases sequenced above minimum read depth, generating more high-quality consensus calls.
GeneRead DNAseq Custom Panel
GeneRead DNAseq Lung Cancer Panel
GeneRead DNAseq Comprehensive Cancer Panel
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GeneRead DNAseq Gene Panel: Performance data
Sample & Assay Technologies Multiplexing Capacity of Target Enrichment
On Popular NGS Platforms
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Sample & Assay Technologies
Specificity (On-target reads)*
Sp
ecif
icit
y(%
on
-tar
get
rea
ds)
No wasting of sequencing capacity
GeneRead DNAseq Custom Panel
GeneRead DNAseq Lung Cancer Panel
GeneRead DNAseq Comprehensive Cancer Panel
* On-Target Reads= Number of reads on target out of total number of reads per run
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GeneRead DNAseq Gene Panel: Performance data
Sample & Assay Technologies DNAseq Gene Panel Application Data
KRAS:G12V is present in all three FFPE lung adenocarcinomas
Detection of Low Prevalence Somatic Mutation in FFPE Lung Adenocarcinoma Sample
Human Lung Cancer GeneRead DNASeq Gene panel was used to enrich 20 genes in genomic DNA isolated from three FFPE lung adenocarcinoma and one FFPE normal lung samples. Sequencing data was analyzed using QIAGEN NGS Data Analysis Web Portal and high quality variants were filtered.
NOTE: KRAS mutations confirmed by either Pyro or ARMS mutation assays (qBiomarker Somatic Mutation PCR Array)
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Sample & Assay Technologies
Where does Library Quantification fit within the NGS workflow?
Next-generation sequencing workflow
GeneRead (DNAseq) Library Quantification and QC System
Sample enrichment
Purificationand
amplification
Library preparation
Next generation sequencing run
Result verification
Why do you want to:1. quantify their NGS DNA libraries2. know the quality of their NGS DNA libraries?
� You have to, in order to ensure high quality reads� Sequencing runs are expensive and time consuming, therefore, you want to ensure that
downstream sequencing analyses are performed on samples of adequate quality for NGS technology.
(Optional)
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Sample & Assay Technologies Why choose qPCR for NGS Library Quantification?
1. BioAnalyzer measures total nucleic acids- Why is this a problem?
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- For each technology, the greater the concentration of each sample (X-axis), the greater the # of clusters
- This should be a linear relationship
- Only qPCR provides this
2. Low limit of detection
3. Consistency of results
Sample & Assay Technologies NGS Library Quantification Made Easy
Adapters can be quantified with qRT-PCR
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Step 4: You perform qPCR with your sample. It should fall between our standard curve created with 5 sequential 10-fold dilutions. THAT is your library concentration to use in preparing your NGS analysis.
Step 1: You have your DNA (whole genome or target enriched)
Step 2: You prepare the DNA library for NGS by adding (via ligation) NGS platform-specific adapters���� NOTE: The adapters are short pieces of DNA
Step 3: We provide you 2 reagents:1. Pre-aliquoted dilutions of the standard 2. qPCR Primer Assays that detect the adapters
Sample & Assay Technologies GeneRead DNAseq Library Quantification Array
Assess target enrichment / sample quality
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Ratio of
Target DNA : Control DNA
Quality of DNA Input to NGS
Sample & Assay Technologies GeneRead Library Quantification System
NGS library quantification for any sequencing application
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, Ion Proton
Sample & Assay Technologies GeneRead DNAseq & LQ System: Summary
� Focused:� Biologically relevant content selection enables deep sequencing on
relevant genes and identification of rare mutations
� Flexible: � Mix and match any gene of interest
� NGS platform independent: � Functionally validated for IT PGM, MiSeq/HiSeq
� Integrated controls:� Enabling quality control of prepared library before sequencing
� Free, complete and easy of use data analysis tool34
Sample & Assay Technologies
Questions?Contact Technical Support: 9 AM – 6 PM M – F ET
Contact: 1-800-742-4368 OR [email protected]
Shankar Sellappan, Ph.D. (Global Product Manager)[email protected]
Ph.D. Trained Application Scientists� Available Before, During, and After Your Experiments for Consultation / Help
GeneRead DNAseq Panel & Library Quant System for NGS
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� Additional NGS Products� GeneRead rRNA Depletion Kit� REPLI-g Mini Kit
� Coming Soon� GeneRead Library Preparation Kits� GeneRead Size Selection Kits
Starter Pack PromotionAvailable to U.S. Customers- Look in email I send to you
Sample & Assay Technologies NOTES / ADDITIONAL INFORMATION
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Sample & Assay Technologies Genomic Information
http://hgdownload.cse.ucsc.edu/downloads.html#human
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Human Genome build 19: hg19
Sample & Assay Technologies FFPE process
Has deamination (C � T and A � G) events during formalin fixation
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Sample & Assay Technologies GeneRead Gene Panel Mastermix
Has HotStart Taq… but not QIAGEN Hotstar Taq DNA Polymerase 203203
It does generate overhang A in PCR products
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Sample & Assay Technologies Species Compatibility
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We currently support human samples with our GeneRead NGS Target Enrichment Panels.
For the LQ kit (the generic version), it could be used on any species, as the adaptors are species independent.
Sample & Assay Technologies Working with UCSC Genome Browser: 2013-01-09
SOP: GeneRead DNAseq Gene Panels with UCSC Browser
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1. Have BED file for gene list with amplicon coordinates2. Go to UCSC Genome Browser: http://genome.ucsc.edu/cgi-bin/hgGateway3. Click: add custom tracks
Sample & Assay Technologies
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4. Upload BED file and hit Submit
5. Click ‘go to genome browser’
Sample & Assay Technologies
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6. Zoom in and out or type in coordinates to move position
NOTE: We need to show what a missing base looks like
Sample & Assay Technologies Concentration Information: Illumina vs IT
Sample & Assay Technologies Mitochondrial Gene Enrichment
Content and Use of controls in LQ
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Since our controls for targeted enrichment are against the genome, and this request is for a ‘custom panel for mitochondrial genes’, is there a point to use our DNAseq version of the library quantification kit if the controls aren’t assessed against mt DNA? And just sell the researcher the generic version of the LQ kit?
I don’t think our controls work on mtDNA, since the copy number ratio of mtDNA:gDNA varies from cell to cell.
13 genes and MT full length designed
Sample & Assay Technologies Multiplexing Samples: Must Index
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Sample & Assay Technologies Your needs
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Your needs
� Simple and easy to perform method, with convenient and high-throughput format
� Fast� Reliable and accurate quantification of sequencing targets� Unmet need: Assess quality of target enriched DNA
� Efficient use of NGS capacity
NGS methodology needs� Too much DNA
� Mixed signals and un-resolvable data� Too little DNA
� Reduced sequencing coverage� Reduced read depth� Empty runs� Increase cost per run� Wasted time
Potential problems in NGS if library not quantified
Potential methods for library QC
� qPCR� Bioanalyzer� Qubit