Transcript - Thermo Fisher Scientific

New flexible solutions for
high throughput genotyping
of CFTR mutations
Toinette Hartshorne, Ph.D.
Senior Staff Scientist
Genetic Analysis Applications
For Research Use Only. Not for use in diagnostic procedures.
The world leader in serving science
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What is Cystic Fibrosis?
•
Cystic Fibrosis is an
autosomal recessive
inherited genetic disease
•
Cystic Fibrosis is the most
common, fatal genetic
disease in European derived populations
1/2500 Caucasian births
•
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High carrier frequency:
approximately 1 in 25
individuals
- Caused by defective CFTR gene: Cystic Fibrosis
Transmembrane Conductance Regulator protein
- Cl- ion transporter
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CFTR Mutation Testing challenges
Commercially available platform limitations
Lack of flexibility in content selection
Fixed panels, some limited to only 23 targets
Limited flexibility in throughput
Maximum of hundred of sample/instrument/day
Long turnaround time
8 to up to 48 hours
High cost per sample
*National Human Genome Research Institute: https://www.genome.gov/10001213
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Platform solution for evaluating CFTR mutations
Various
Sample Types
KingFisher™
& MagMAX™
DNA
Extraction
QuantStudio™
Systems
Flexible
Formats
Flexible, Scalable, Low cost
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TaqMan™
Assays
TaqMan Assay Technology
Gold Standard technology - Universal PCR protocol
- 4 hours PCR reaction with OpenArray plate format
- 2 hour PCR reaction with 96- and 384-well plate formats
Broad assay collection available
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7 Million Predesigned TaqMan SNP Genotyping Assays
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2,700 TaqMan Drug Metabolism Assays
•
200 Qualified CFTR TaqMan Assays
Custom TaqMan Assays
Most common CFTR mutations around the world
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Design custom panels from a menu of 198 CFTR targets
Majority coverage of CFTR2 mutation list
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CFTR Qualified Assays Performance Testing
Designed and tested >400 unique assays to ~200 CFTR targets
Key mutations: ACMG panel, CFTR2, collaboratations, mutation frequency
Tested assays with 47 Coriell gDNAs with known CFTR genotypes
384-well and OpenArray plates on QuantStudio 12K Instrument
Tested candidate assays with synthetic templates
Normalized homozygous & heterozygous plasmid controls
Evaluated performance and concordance metrics
Repeatability and Reproducibility studies
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gDNAs samples: cell line, blood, buccal
High Performance Results with TaqMan OpenArray
and 384-well Plates
Open Array
384-well
Lot 1
Lot 2
Lot 3
NA
Call Rate
99.46%
99.56%
99.76%
99.90%
Accuracy
99.97%
99.98%
99.97%
100.00%
Data points
46,638
14,346
16,280
12,880
3659delC
F508del
N1303K
W1282X
Representative Data Shown
Study included 64 Whole Blood gDNA samples: 28 CF Positive Samples & 36 WT Samples
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CFTR TaqMan SNP Genotyping Assays set
• 200 assays to 198 CFTR mutation targets
• Challenging targets genotyped with non-standard TaqMan assays:
Assays that overlap another mutation, e.g. F508del region
Homopolymer mutations, e.g. 2184insA region
Poly T tract 5T/7T/9T polymorphism
Large deletions
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Clustered mutation targets: F508del region assays
F508del assay probes overlap I506V, I507del, I507V, F508C
ACCATTAAAGAAAATATCAT[-/CTT]TGGTGTTTCCTATGATGAAT
Assay probes for these clustered
mutations specifically identify their
mutation target, and fail to bind
amplicons containing a nearby,
underlying non-target mutation.
e.g. F508del mu/mu samples are
not detected by assays to the 4
nearby variants.
(E)
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Control Coriell gDNA samples run on OpenArray plates
Homopolymer targets: 2184delA region assays
2184delA
c.2052delA
p.Lys684AsnfsX38
CTGTCTCCTGGACAGAAACAAAAAA[A/-]CAATCTTTTAAACAGACTGGAGAGT
2183delAAc.2051_2052delAA
p.Lys684ThrfsX4
CTGTCTCCTGGACAGAAACAAAAA[AA/-]CAATCTTTTAAACAGACTGGAGAGT
2183AA->G
c.2051_2052delAAinsG
p.Lys684SerfsX38
CTGTCTCCTGGACAGAAACAAAAA[AA/G]CAATCTTTTAAACAGACTGGAGAGT
2184insA
c.2052_2053insA
p.Gln685ThrfsX4
CTGTCTCCTGGACAGAAACAAAAAAA[-/A]CAATCTTTTAAACAGACTGGAGAG
ACMG panel (7 > 6 As)
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Homopolymer indels are usually not amenable to TaqMan assay design
PolyT tract 5T/7T/9T assays and data analysis
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The 5T/7T/9T alleles are a component of the intron 8 polyAG/polyT mutation
5T affects CFTR RNA splicing, resulting in low levels of CFTR protein
• 5T is incompletely penetrant; impact is most severe when in cis with R117H
5T
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Genotype
5T assay result
7T (TT) = VIC / 5T (-) =FAM
9T assay result
9T (TT) =VIC / 7T (-) = FAM
5T/5T
-/-
-/- , und, or noamp
5T/7T
TT/-
TT/TT
5T/9T
TT/-
TT/TT, und
7T/7T
TT/TT
-/-
7T/9T
TT/TT
TT/-
9T/9T
TT/TT
TT/TT
9T
Whole blood gDNA samples run on OpenArray plates
Large deletions: CFTRdele2,3 & CFTRdele22,23
CFTRdele2,3 wild type
CFTRdele2,3 mutation
CFTRdele2,3 het
CFTRdele2,3 het
Manually called as ‘no amp’
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CFTRdele2,3 and CFTRdele22,23 large deletions are interogated by a pair of
modified copy number assays:
• Wild type (wt) and mutant (mu) allele assays are run separately.
• Each assay contains one functional probe (wt = VIC; mu=FAM)
Heterozygous samples amplify with both assays; homozygous wild type
samples only amplify with the wild type assay.
Control Coriell gDNA samples run on OpenArray plates
Control kit available with full coverage of all 200 qualified
CFTR TaqMan Assays
CFTR TaqMan Assay Controls Kit
• Contains 18 control plasmids, 3 kb on average
• Each plasmid contains heterozygous sequences for 10 to 14 targets
• Recommend testing plasmid controls with wild type gDNA
3659delC
F508del
N1303K
CFTR synthetic controls used to generate HET mutation results
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W1282X
Sample-to-answer workflow for CFTR mutation testing
Genotyping Workflow
384-well Plates
OR
Sample Type:
KingFisher Flex System
with MagMax kits
Whole blood
Buccal Swab
CFTR
mutation
Results
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Genotyping data for
rare allele detection*
* TaqMan Genotyper™ Software or Thermo Fisher Cloud GT App
OpenArray
AccuFill System
for sample loading
Run Assays on
QuantStudio
System
QuantStudio 12K Flex
Enables Breadth in Content and Format Flexibility
Choice of 4 formats
Individual Assays in Tubes: Flexible, any instrument
TaqMan Assay Plates – FAST & Standard
96 well & 384 well optical plates
Custom plating of assays
TaqMan Arrays
384 well microfludic cards
Custom assay panels
TaqMan OpenArray Plates
3,072 well array plate; highest throughput
Lowest cost per rxn
• Approximately 1-2 minutes to exchange the block / heated cover / plate holder.
• No re-calibration required
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Qualified CFTR TaqMan Assay offering
Key benefits
Flexible content
Custom panels from 24 to 180 assays per plate
Flexible throughput
From 6 to 576 samples/instrument/day
Complete workflow
From blood/buccal cell sample to genotype
Low cost per sample
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Poster P01.020D
Monday afternoon
Real-Time PCR Using OpenArray® Technology
http://www.thermofisher.com/CFTR
For Research Use Only. Not for use in diagnostic procedures.
© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of
Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a
registered trademark of Roche Molecular Systems, Inc., used under permission and license.
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OpenArray Plate Technology
33 nL
Hydrophilic
TaqMan SNP
Genotyping Assays
Hydrophobic
Number of
Samples
26
X
96
60
X
48
120
X
24
180
X
16
240
X
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Flexibility to customize your own panels
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