The document describes QIAGEN's GeneRead DNAseq Targeted Exon Enrichment and GeneRead Library Quantification System for next generation sequencing. It discusses targeted enrichment workflow and principles, data analysis, pathway content of panels, performance data and application examples. It also covers the library quantification workflow, using qPCR to quantify sequencing libraries, and a DNAseq library quantification array to assess sample quality. The document is aimed at promoting these NGS sample preparation and analysis solutions to potential customers.
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Complete Biological Story
Built on Pathway / Network Analysis
Angiogenesis
Inflammation
Pathway
Cell cycle
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Patient sample =
Wild-type =
Determining DNA Differences
ATGCCATCTGGGACGGGTCAGTAG
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 =
Amino Acid Sequence =
ATG CCA TCT GGG ACG GGT CAG TAG
Met P S G T G Q Stop
Valine
Glycine
Compare the two amino acid sequences:
Patient sample AA Sequence =
Wild-Type AA Sequence =
Met P S G T G Q Stop
Met P S V T G Q Stop
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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What is NGS (Next Generation Sequencing)?
Massively Paralleled Sequencing
Instead of sequencing a DNA sequence from
Sequence many small pieces at the same time
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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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Your NGS research needs
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
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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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New NGS Workflow with Targeted Enrichment
80 ng
Achieve more sensitive mutation detection
with 1 additional step
• 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
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Target Enrichment: Principles
Multiplex PCR-enabled enrichment of gene of interest
We provide primer sets that produce overlapping PCR products
• For any gene or set of genes in the human genome
Division of non-adjacent gene primer sets into
4 tubes decreases non-specific amplification.
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Complete Analysis Workflow
With integrated controls to assess sample quality / TE process
Coming Soon!
-
QIAGEN GeneRead Library Preparation Kits (for IT and IL)
QIAGEN GeneRead Size Selection Kits
QIAGEN GeneRead Library Indexing Kits
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QIAGEN NGS Platform
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NGS Sequence Analysis and Variant ID Software
FREE Complete & Easy to use Data Analysis with Web-based Software
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Read Mode: Paired End vs Single End
Goal: Increased # of Reads / Amplicon
Sequence of Interest:
TACGCATCGATGCGGTAACTGCTGATCGTCGTAGTGCTAGCTGA
Single End Sequencing:
TACGCATCGATGCGGTAACTGCTGATCGTCGTAGTGCTAGCTGA
Paired End Sequencing (both ends are sequenced):
TACGCATCGATGCGGTAACTGCTGATCGTCGTAGTGCTAGCTGA
AGTCGATCGTGATGCTGCTAGTCGTCAATGGCGTAGCTACGCAT
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How Genes on Panels Are Selected
Comprehensive Cancer Panel (124 genes)
Disease Focused Gene Panels (20 genes)
Breast cancer
Liver cancer
Colon Cancer
Lung Cancer
Gastric cancer
Ovarian Cancer
Leukemia
Prostate Cancer
Genes with High Relevance
Biologically relevant gene content
Clinically relevant: Published association with the
disease state
– Multiple Publically accessible databases
– Text mining tools
– Manually curated
Genes Involved in Disease
ALSO AVAILABLE: Custom Panels from ANY GENE
or COLLECTION OF GENES in Human Genome
Technically relevant gene content
Most frequently mutated genes
Specific feedback from the thought leaders
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Targeted Enrichment: Panel Information
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NGS Target Enrichment Design Strategy
Commonly encountered issues solved by GeneRead Algorithm
•
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
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Design Coverage Information Provided
Base Pairs Covered
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BED File Information
M1 = Covered
Regions
M*1 =
Uncovered
Regions
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Gene Visualization with UCSC Genome Browser
Instructions will be provided online at SABiosciences.com
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GeneRead DNAseq Gene Panel: Performance data
% Covered by Design
Design coverage
GeneRead DNAseq
Custom Panel
GeneRead DNAseq
Lung Cancer Panel
GeneRead DNAseq
Comprehensive Cancer Panel
Discover more potential variants by covering more exons
for genes of interest in assay design
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GeneRead DNAseq Gene Panel: Performance data
Sequence coverage uniformity
GeneRead DNAseq
Custom Panel
GeneRead DNAseq
Lung Cancer Panel
GeneRead DNAseq
Comprehensive Cancer Panel
More bases sequenced above minimum read depth,
generating more high-quality consensus calls.
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Multiplexing Capacity of Target Enrichment
On Popular NGS Platforms
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GeneRead DNAseq Gene Panel: Performance data
Specificity
(% on-target reads)
Specificity (On-target reads)*
GeneRead DNAseq
Custom Panel
GeneRead DNAseq
Lung Cancer Panel
GeneRead DNAseq
Comprehensive Cancer Panel
No wasting of sequencing capacity
* On-Target Reads= Number of reads on target out of total number of reads per run
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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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Next-generation sequencing workflow
Where does Library Quantification fit within the NGS workflow?
Purification
and
amplification
Sample
enrichment
Library
preparation
Next generation
sequencing run
Result
verification
(Optional)
GeneRead (DNAseq) Library
Quantification and QC System
Why do you want to:
1. quantify their NGS DNA libraries
2. 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.
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Why choose qPCR for NGS Library Quantification?
1. BioAnalyzer measures total nucleic acids
- Why is this a problem?
2. Low limit of detection
3. Consistency of results
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For each technology, the greater the
concentration of each sample (X-axis),
the greater the # of clusters
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This should be a linear relationship
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Only qPCR provides this
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NGS Library Quantification Made Easy
Adapters can be quantified with qRT-PCR
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 platformspecific 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
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.
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NGS Library Quantification Made Easy
Adapters can be quantified with qRT-PCR
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 platformspecific 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
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.
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GeneRead DNAseq Library Quantification Array
Assess target enrichment / sample quality
Ratio of
Target DNA : Control DNA
Quality of DNA Input to
NGS
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GeneRead Library Quantification System
NGS library quantification for any sequencing application
, Ion Proton
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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 tool
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GeneRead DNAseq Panel & Library Quant System for NGS
Additional NGS Products
GeneRead rRNA Depletion Kit
REPLI-g Mini Kit
Coming Soon
GeneRead Library Preparation Kits
GeneRead Size Selection Kits
Starter Pack Promotion
Available to U.S. Customers
- Look in email I send to you
Questions?
Contact Technical Support: 9 AM – 6 PM M – F ET
Contact: 1-800-742-4368 OR support@SABiosciences.com
Shankar Sellappan, Ph.D. (Global Product Manager)
Shankar.Sellappan@QIAGEN.com
Ph.D. Trained Application Scientists
Available Before, During, and After Your Experiments for Consultation / Help
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NOTES / ADDITIONAL INFORMATION
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Genomic Information
http://hgdownload.cse.ucsc.edu/downloads.html#human
Human Genome build 19: hg19
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Species Compatibility
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.
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Working with UCSC Genome Browser: 2013-01-09
SOP: GeneRead DNAseq Gene Panels with UCSC Browser
1. Have BED file for gene list with amplicon coordinates
2. Go to UCSC Genome Browser: http://genome.ucsc.edu/cgi-bin/hgGateway
3. Click: add custom tracks
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