SNP genotyping on qPCR platforms: Troubleshooting for amplification and cluster separation

SNP genotyping on qPCR platforms:
Troubleshooting for amplification and cluster separation
Daniel Tsang, Research Scientist
1

• Background: Genotyping by qPCR
• Introduction of rhAmp™ SNP Genotyping System
• Common genotyping failure modes and solutions
– Benefits of IDT rhAmp SNP Genotyping System
2
Outline: SNP genotyping on qPCR platforms

• Introduction of rhAmp SNP Genotyping System
3

Introduction to genotyping by qPCR
• Technologies used for genotyping
– Sequencing: Discovering novel SNPs
• Large number of SNPs
• Small number of samples
– Array: Detecting known SNPs
• Large number of SNPs
• Small number of samples
– Quantitative PCR (qPCR): Detecting known SNPs
• Fewer number of SNPs
• Large number of samples
• Small quantity of samples
• Shorter workflow
4
Number of samples
Numberoftargets
Seq
Array
qPCR

Factors to consider for high confidence calls
• Lack of amplification in
no template control (NTC)
reaction
• High signal-to-noise ratio
• Tight clusters
• Clear cluster separation
5
Example of allelic discrimination (AD) plot
Yakima Yellow is a registered trademark of Elitech Group.

6

rhAmp SNP Genotyping mechanism
• Step 1: Primer design and annealing
– Modified primers include a 3′ blocking group and an RNA base next to the SNP site.
– Primers must be annealed to the matched target for RNase H2 cleavage to occur, resulting in deblocking of primers.
• Step 2: Primer cleavage and extension
– Primer cleavage by RNase H2 generates a 3′-OH group extendable by the highly discriminating, mutant Taq polymerase.
• Step 3: Universal reporter incorporation
– rhAmp primers have 5′ tails, which incorporate a universal 5′ nuclease assay reporter system.
• Step 4: Probe cleavage and signal generation
– The FAM and Yakima Yellow reporter dyes distinguish the 2 alleles.
7

Complete rhAmp SNP Genotyping System
• Simple, one-tube chemistry works on all common qPCR platforms
• >10 million predesigned assays for human SNPs
• High performance, custom assay design tool for other species
8
1. Select your assay type
2. Select your species
3. Select your target input
format
4. Copy and paste your
target list

9

Common failure modes in SNP genotyping by qPCR
1. NTC amplification
2. No amplification
3. Weak amplification
4. Merging clusters
5. Single cluster with no call
6. Trailing clusters
7. More than 3 clusters
10

Importance of having no template control reactions
• NTCs serve as reference points for genotyping
11
1.2 1.7 2.2 2.7 3.2 3.7 4.2
Allele 1 (FAM Rn)
2.8
2.3
1.8
1.3
Wrong genotype assignment without NTCs Correct genotype assignment with NTCs
2.8
2.3
1.8
1.3
1.2 1.7 2.2 2.7 3.2 3.7 4.2
Allele 1 (FAM Rn)
Allele2(YakimaYellowRn)

Importance of having no template control reactions
• Use of NTCs is recommended to confirm manual genotype calls
• NTCs help rule out non-specific amplification
12
Correct genotype assignment
with no NTC amplification
No genotype assignment
with NTC amplification
NTCs NTCs
Allele2(x103YakimaYellowRFU)
Allele 1 (x103 FAM RFU)Allele 1 (x103 FAM RFU)

• AD plot characteristics
– NTC signals are high
– NTCs are close to sample data points
– Clusters merge or have no separation
• Results
– Wrong genotype call
– No call
NTCs
Samples
indicated as no
amplification
Allele 1 (x103 FAM RFU)
13

14
DNA
contamination in
NTC
Primer-dimers
Extensive
post-PCR
hold time
Sample wells
incorrectly
assigned as “NTC”
Use new reagents
Contact technical
support for
re-design
Add enzyme
heat-kill step
Re-assign “NTC”
in software
Causes
Solutions
Instrument / software Sample Reagent Assay

15
Homo1 cluster is close to Het cluster,
NTC positive amplification
1. NTC amplification—solution
Homo1 cluster is further away from Het cluster,
No NTC amplification
NTCs
NTCs
Allele 1 (x103 FAM RFU) Allele 1 (x103 FAM RFU)
• Fewer PCR cycles reduce NTC amplification
PCR cycles
decreased from
45 to 40 cycles

2. No amplification
– Sample signal is very low
– NTC data is close to sample data
– No cluster separation
– Dispersed clusters
• Results
– No call
16
NTCs
Allele2(YakimaYellowRFU)
Allele 1 (FAM RFU)

2. No amplification
17
Poor sample
quality
Incorrect dilution
of assays
Enzyme is not
active
Assign wrong
reporter dye in
software
Use new samples
Repeat experiment
at correct assay
dilution
Check thermal
cycling protocol
Check run file and
re-assign reporter
dye
Causes
Solutions

3. Weak signal from one of the reporter dyes
– No NTC amplification
– Two clusters compress together
– One dye has strong signal, but
the second dye has weak signal
• Results
– Correct genotype call with low
confidence
– No call
0.8
0.6
0.4
0.2
0.0
Allele2(YakimaYellowdRn)
0.3 0.8 1.3 1.8 2.3 2.8
Allele 1 (FAM dRn)
Weak Allele 2 signal
18

3. Weak signal from one of the reporter dyes
19
Presence of PCR
inhibitors
Amplification
biased to one
primer
PCR cycle number
is too low
Perform sample
titration
Contact technical
support for
re-design
Increase PCR
cycle number
Causes
Solutions
Instrument / software Sample Assay

3. Weak signal from one of the reporter dyes—solution
DNA input
increased from
1 ng to 3 ng
• Increased DNA input improves cluster separation
20

Improved cluster separation with rhAmp SNP Genotyping System
rhAmp SNP Genotyping System
• NTC amplification is minimal, and NTC location is consistent
• Robust, consistent signal improves cluster separation
• Greater cluster separation provides higher confidence for
automated and manual calls 21
AD plots for 18 assays combined
5′ nuclease genotyping assayrhAmp SNP Assay

4. Clusters merge together
– Clusters merge together
– High signals
• Results
confidence
– No call
0.0 1.0 2.0 3.0 4.0 5.0 6.0
Allele 1 (FAM dRn)
3.7
2.7
1.7
0.7
0.2
No cluster separation
22

4. Clusters merge together
23
DNA sample input
is too high
Non-specific
amplification from
the other primer
Extensive
post-PCR
hold time
Too many PCR
cycles
Reduce DNA input
amount
Contact technical
support for
re-design
Add enzyme
heat-kill step
Reduce PCR
cycle number
Causes
Solutions

4. Clusters merge together—solution
Sample input
decreased from
10 ng to 3 ng
• Decreased DNA input improves cluster separation
24

25
– Single cluster
– One dye has strong signal, but the
second dye has weak signal
4.0
3.5
3.0
2.5
2.0
1.5
1.0
1.0 2.0 3.0 4.0 5.0 6.0
• Result
– No genotype call
Allele 1 (FAM Rn)
5. Single cluster

5. Single cluster
26
Assay with low
minor allele
frequency (MAF)
Software cannot
generate
automated call
Run positive
controls with all
three genotypes
Causes
Solutions
One allele-specific
primer does not
work
Contact technical
support for
re-design
Instrument / software Assay
Run positive
controls with all
three genotypes
Assay

gBlocks® Gene Fragments are available as control templates
for each rhAmp SNP assay
• To order gBlocks fragments, visit www.idtdna.com/gBlocks
27

Allele 1 (FAM Rn)
Positive controls improve genotype calling
28
CYP2C9 (rs72558187)
10 ng of human gDNA
purified from whole blood
Analysis including 1000 copies of
gBlocks controls for each genotype
• Manual and automated calls are improved with positive controls for
each genotype cluster
4.0
3.5
3.0
2.5
2.0
1.5
1.0
1.0 2.0 3.0 4.0 5.0 6.0
Allele 1 (FAM Rn)
4.0
3.5
3.0
2.5
2.0
1.5
1.0
1.0 2.0 3.0 4.0 5.0 6.0
gBlocks controls

– No clear separation between
clusters and NTC
– Weak signal for some samples
with no genotype call
– Samples that drifted out of cluster
may have no genotype call
• Results
– No call for many samples1.1 1.6 2.1 2.6 3.1
Allele 1 (FAM Rn)
4.0
3.5
3.0
2.5
2.0
1.5
1.0
29

30
Broad range of
DNA sample
concentrations
Low PCR
efficiency
Low master mix
concentration
Wrong cycling
conditions
Normalize
DNA samples
Contact technical
support for
re-design
Check experiment
protocol
Check cycling
conditions
Causes
Solutions

7. Multiple clusters (more than 3 clusters)
31
– More than 3 clusters
– Weak signal for some samples
– Samples that drifted out of cluster may
have no genotype call
• Results
confidence
– No call
0.2 0.7 1.2 1.7 2.2 2.7 3.2 3.7
Allele 1 (FAM dRn)
3.3
2.8
2.3
1.8
1.3
0.8
0.3

7. Multiple clusters (more than 3 clusters)
32
Copy number
variation
SNP in primer
binding region
Assigned wrong
assay ID to wells
Sequence samples
to determine copy
number variation
Design primers at
a different location
Re-assign
assay ID
Causes
Solutions
Instrument / software Sample Assay

Take home messages
• rhAmp SNP system offers multiple
advantages for genotyping
– Clean NTCs
– Robust and consistent signals
– Improved cluster separation
– gBlocks Gene Fragments are available
as control templates for each assay
34
4.0
3.5
3.0
2.5
2.0
1.5
1.0
1.0 2.0 3.0 4.0 5.0 6.0
gBlocks controls
More information at:
www.idtdna.com/rhAmp-Genotyping
Webinar:
www.idtdna.com/video
Allele 1 (FAM Rn)

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SNP genotyping on qPCR platforms: Troubleshooting for amplification and cluster separation