2012 10-24 - ngs webinar

Sample & Assay Technologies

GeneRead DNAseq Targeted Exon Enrichment &
GeneRead Library Quantification System for
Next Generation Sequencing
Shankar Sellappan, Ph.D.
Global Product Manager
Shankar.Sellappan@QIAGEN.com
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Pathway-Focused Analysis Tools

2


GeneRead DNAseq & Library Quant NGS System

Agenda
 Introduction
 Targeted Enrichment
 Workflow
 Principles
 Data Analysis
 Pathway Content
 Performance Data
 Application Example
 Library Quantification
 Workflow
 Application Example
 Summary
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Individual
participants

Cell cycle

P53
MDM2
Cyclins
CDKs

Biological Markers Define Biological Processes

Angiogenesis

VEGF
bFGF
ANGPT
PDGF

Inflammation

IL8
TLR
IFNγ
TNFβ

4


Complete Biological Story
Built on Pathway / Network Analysis

Angiogenesis

Inflammation

Pathway

Cell cycle

5


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.

8


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

9


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 increases amplification specificity.
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Targeted Enrichment: Details

~ 150 bp PCR
products

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Complete Analysis Workflow
With integrated controls to assess sample quality / TE process

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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:
Paired End Sequencing (both ends are sequenced):

AGTCGATCGTGATGCTGCTAGTCGTCAATGGCGTAGCTACGCAT


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


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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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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Sequence coverage uniformity

GeneRead DNAseq
Custom Panel

GeneRead DNAseq
Lung Cancer Panel

GeneRead DNAseq

More bases sequenced above minimum read depth,
generating more high-quality consensus calls.
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Specificity
(% on-target reads)

Specificity (On-target reads)*

GeneRead DNAseq
Custom Panel

GeneRead DNAseq
Lung Cancer Panel

GeneRead DNAseq

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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Your needs

Potential problems in NGS if library not quantified
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
Your needs
 Simple and easy to perform method, with convenient and highthroughput format
 Fast
 Reliable and accurate quantification of sequencing targets
 Unmet need: Assess quality of target enriched DNA
 Efficient use of NGS capacity
Potential methods for library QC
 qPCR
 Bioanalyzer
 Qubit
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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
-

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
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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 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

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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2012 10-24 - ngs webinar

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2012 10-24 - ngs webinar