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HORIZON DISCOVERY
Molecular QC: Using Reference Standards in NGS Pipelines21st May 2015
Jonathan Frampton, PhD and Natalie LaFranzo, PhD
3
Clinical Application of Next Generation Sequencing
Using just one sample, one workflow can test for mutation status across multiple genes
External Quality Assessment
5
T790M &
L858R
E746_A
750del
Wild
type
Wild
type
E746_A
750del
T790M &
L858R
E746_A
750del
Wild
type
T790M &
L858R
T790M &
L858R
E746_A
750del
T790M &
L858R
E746_A
750del
Wild
type
Wild
type
Wild
type
Wild
type
G719S
T790M &
L858R
G719S0
5
10
15
20
25
30
35
40
EGFR Genotyping ErrorsExternal Quality Assessment 2014
EGFR Sample Tested
Perc
enta
ge o
f Inc
orre
ct R
esul
ts
European Molecular Quality Network (EMQN)
6
For Research Use Only
Next-Generation Sequencing Introduction
Also known as high-throughput or massively-parallel sequencingโข Allows us to address questions that require a lot of data
โข Has been applied to scientific questions across industriesโข Pharma โข Biotechโข Biofuelsโข Agricultureโข Food Scienceโข Archeologyโข Medicineโข โฆ
vs.
7
For Research Use Only
Next-Generation Sequencing Introduction
DNA de novo
assembly
DNAresequencing
DNAepigenomics
RNAtranscriptomics
DNAmetagenomics
And moreโฆ
8
For Research Use Only
Next-Generation Sequencing Introduction
DNA de novo
assembly
DNAresequencing
DNAepigenomics
RNAtranscriptomics
DNAmetagenomics
And moreโฆ
9
For Research Use Only
Patient-derived Samples โ DNA Analysis
Unique challenges:
โข Heterogeneous
โข Low quantity
โข Poor quality
โข Low-allelic frequency detection desired
13
For Research Use Only
DNA Extraction Variability from FFPE
Promega
Max
well (n=12)
Promega
Mag
nesil (n
=6)
Promega
ReliaPrep (n
=6)
Qiagen Dneasy
(n=6)
Roche Cobas
(n=6)0%
10%20%30%40%50%60%70%
DNA Recovery from Total Theoretical Yield
Extraction Kit
Perc
enta
ge D
NA
Rec
over
ed
In house generated data set.
15
For Research Use Only
DNA Quantification
Lab A
Lab B
Lab C
Lab D
Lab E
Lab F
Lab G
Lab H
Lab I
Lab J
Lab K
Lab L
Lab M
Averag
e
Median
0
5
10
15
20
25
30
35
40Nanodrop:Qubit (N/Q Ratio)
Participant Laboratory
N/Q
Kapp J R et al. J Clin Pathol doi:10.1136/jclinpath-2014-202644
17
For Research Use Only
Sequencing Library Preparation
Enrichment options:
โข whole-genome (not enriched)
โข whole-exome capture
โข custom capture
โข capture-based panels
โข off-the-shelf amplicon panels
โข custom amplicon panels
Goal: Use a reference standard that reflects your actual sample.
19
For Research Use Only
Variability
Platform: QX100โข Droplet Digital PCR
Ampliseq Cancer Hotspot Panel v2
(Average of 8 runs)Ampliseq Cancer Hotspot Panel v2
Ampliseq Cancer Hotspot Panel v2
Sequencing Depth: N/A N/A 2000x 3000-4000x Average 5000x
Gene Variant Specification In house validation Partner A Partner B Partner C
BRAF V600E 10.5 10.2 10.3 9.9 9.1
KIT D816V 10.0 10.4 10.1 10.0 11.0
EGFR ฮE746 - A750 2.0 2.0 Not detected 2.3 Not detected
EGFR L858R 3.0 2.7 2.4 2.7 2.1
EGFR T790M 1.0 0.9 Not detected 0.8 Not detected
EGFR G719S 24.5 24.4 24.8 23.7 23.1
KRAS G13D 15.0 16.1 15.5 16.3 12.4
KRAS G12D 6.0 5.0 5.1 5.2 Not detected
NRAS Q61K 12.5 12.8 12.6 9.0 12.7
PIK3CA H1047R 17.5 18.6 17.9 16.7 16.8
PIK3CA E545K 9.0 8.9 8.8 3.2 8.4
23
For Research Use Only
Theoretical Limit of DetectionDependent on:
(1) molecular uniqueness/deduplication, (2) input, and (3) coverage
Reference Sequence
24
For Research Use Only
Theoretical Limit of Detection
4๐๐๐๐๐ h๐ค๐๐ก ๐๐ข๐ก๐๐ก๐๐๐๐๐ ๐๐๐ก๐๐๐๐ ๐ก3333๐๐๐๐๐ (๐๐๐๐๐๐ข๐๐๐ )
=0.12% ๐๐๐๐๐๐๐ ๐๐๐๐๐ข๐๐๐๐ฆ
Reference Sequence
For 10 ng input amount (or 3333 molecules):
25
For Research Use Only
Theoretical Limit of Detection
Reference Sequence
For high input: 4๐๐๐๐๐ h๐ค๐๐ก ๐๐ข๐ก๐๐ก๐๐๐๐๐ ๐๐๐ก๐๐๐๐ ๐ก
ยฟ๐๐๐๐๐ =๐ฟ๐๐๐๐ก ๐๐ ๐๐๐ก๐๐๐ก๐๐๐
4๐๐๐๐๐ h๐ค๐๐ก ๐๐ข๐ก๐๐ก๐๐๐๐๐ ๐๐๐ก๐๐๐๐ ๐กยฟ๐๐๐๐๐
=0.01% ๐๐๐ ๐๐๐๐
# reads = 40,000x!
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For Research Use Only
Actual Limit of Detection
Target Allelic Frequency
50% Mutant DNA (orange lid)
Wild Type DNA (white lid)
20% 4ยตl 6ยตl
10% 2ยตl 8ยตl
5% 1ยตl 9ยตl
1%1ยตl of prepared 10%
dilution 9ยตl
0% 0ยตl 10ยตl
Total Volume 7ยตl 42ยตl
27
For Research Use Only
Actual Limit of Detection
0% 5% 10% 15% 20% 25%0%
5%
10%
15%
20%
25%Limit of Detection Results
Target Allelic Frequency
Aver
age
of A
ctua
l Alle
lic
Freq
uenc
y D
etec
ted
Alternative: Tru-Q Multiplex standards available at 5%, 2.5% and 1.25% allelic frequencies covering multiple mutants.
1% not detected
28
For Research Use Only
Exciting Challenges
HD701 vs. HD751 vs. HD200
โข Multiple formats for Quantitative Multiplex Reference Standardโข 11 validated positive mutationsโข Frequency range: 24%-1%
โข HD701 โhigh molecular weight DNA extracted directly from cells
โข HD751 โ formalin-compromised DNA (harsh formalin treatment, highly degraded)โข Lanes 3 and 5 on right
โข HD200 โ mild-formalin fixation, embedded in paraffin (FFPE format), once extracted shows little degradationโข Lanes 2 and 5 on right
Genomic DNA Tapescreen assay
[bp] 1 2 3 4 5
31
For Research Use Only
How to Test the Robustness and Sensitivity of your Workflow and Assay
Sensitivity of your Assay
HD701
Formalin Intensity
HD200
Robustness and Sensitivity of your Workflow
HD-C751
FFPE
DNA
Robustness of your Assay
HD-C749
GIAB FFPE Samples
32
Routinely monitor the performance of your workflows and assays with independent external controls
What extraction and quantification methods are you
using?
What is the limit of detection of your
workflow?
Is the impact of formalin treatment interesting to you?
What is the impact of assay failure in your laboratory and how do you monitor for it?
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