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Quantitative Real-time PCR (qPCR)

Presented by Solmaz Oskooei

Jenny Carter, Kamila Jagiello, Rubin Wang

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What is qPCR? What do we use it for at GOSH? How does it work? What can go wrong? Recent Cases NGS follow up

Introduction

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What is qPCR?

Quantitative real-time PCR

Measurements of fluorescence are

made in real time during the

exponential phase

Determines quantity of an amplified

product (copy number).

Introduction

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What do we use qPCR for in the laboratory?

1. To confirm a microarray result

(gain or loss of copy number)

2. To determine inheritance (testing parents)

3. Further investigation of a potential

copy number change found via NGS

4. NIPD – fetal sexing

Introduction

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How does it work?

Design primers for a ~100bp region of interest

Measure patient and control DNA to ensure all at the same

starting concentration and amount.

Add primers and Master mix including

SYBR Green.

(can use Taqman but SYBR green

is cheaper and faster!)

Introduction

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How does it work?

• PCR takes place along with melt curve analysis all in one reaction

• Measurements of fluorescence are taken in real time.

• Amount of amplified product from patient is compared to control = relative

quantitation.

• Results normalised using endogenous controls (VCPIP1 and MANEA)

• A stunning bar graph is produced.

Introduction

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What is the difference between QF-PCR (quantitative fluorescence PCR) and qPCR?

Introduction

qPCR

-Non labelled primers -Real time measurements of quantity -“Bespoke” primer design

QF-PCR -Labelled primers -Utilises STRs

-Can include several probes in one assay

Both can be

used to

determine copy

number

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Neither of these are the same as qPCR………

Introduction

qRT-PCR

Quantitative reverse-transcriptase PCR

RT-PCR -Reverse transcriptase PCR

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Primer

design

Primer Design

Primer

Testing Reporting qPCR

Analysis/

Checking

Good design is crucial as qPCR is VERY sensitive.

Use several resources :

- Alamut

- ENSEMBL

- Primer-BLAST

- gnomAD

- OligoCalc

- UCSC insilico PCR

95-125bp amplicon

Two pairs for every family

Details entered onto primer database (1870 primers so far!)

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Primer Testing

All new primers are tested by running a qPCR with and without control

DNA. This is to check for primer specificity.

Can test 16 primer pairs per plate

Discarded if fail primer testing

Primer

design

Primer

Testing Reporting qPCR

Analysis/

Checking

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Primer Testing

Blank (NTC)

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Setting Up a qPCR

Once a suitable primer has been identified for a family, a worksheet is

produced.

One 96 well plate will contain:

Two families (total of up to 6 patients)

Two normal DNA control samples (one male, one female)

Two control primers (endogenous controls MANEA and VCPIP1)

Blank (NTC) control wells for all 4 primers

Primer

design

Primer

Testing Reporting qPCR

Analysis/

Checking

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Setting Up a qPCR

1. DNA Quantification

Getting the same starting DNA concentration is critical!!

300ng DNA per patient

2. Primer Preparation

Primers are added to tubes containing the Power SYBR green master mix.

3. Loading DNA + Primer

Then divide each DNA/primer mix between three wells on 96 well plate.

Primer

design

Primer

Testing Reporting qPCR

Analysis/

Checking

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Analysis

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Analysis

Relative Quantification

Compare the RQ (Relative Quantification) value for each patient against a

normal control

Primer

design

Primer

Testing Reporting qPCR

Analysis/

Checking

RQ range

Normal 0.8 - 1.2

Gain >1.3

Loss <0.7

Inconclusive 0.71 - 0.79 or 1.21-1.29

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Analysis

Proband Mother Control1 Control2

Proband Mother Father Control1 Control2

Inherited gain

Inherited

loss

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Advantages

Can be used to detect any copy number changes (depending on

location), down to a minimum of 100bp.

Relatively inexpensive

Primers can be designed and ordered relatively quickly and

easily

Targeted test – avoids incidental findings

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Limitations

May not be possible to design a primer

Preparation and set up is labour intensive (2-3 hours)

Low throughput as can only do two families per plate, maximum of two

plates per day, per machine.

No positional information for duplications.

Very sensitive – technically challenging, precise pipetting essential.

Cannot detect mosaicism

Cannot determine size of copy number loss

Does not give an integer copy number value.

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What can go wrong?

Problem Solution

Primer design problems: e.g. SNPs,

amplification in blank wells, haplotype,

pseudogenes

• Re-design and repeat with new primers.

• Use SNP-free controls (not available for

microarray follow ups)

• use two sub-optimal primers together

• use a different technique (FISH/array)

Contamination in blank wells repeat and discard working solutions

High SD repeat

Discrepant controls discard controls and repeat with different

controls. Check to make sure ROI is not in a

variable region!

Inconclusive result repeat

Variation in normal population Do not follow up

DNA degradation Request another sample

Repeat so much that run out of DNA Request another sample

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Therefore even if a primer has worked well previously – it still may take

several attempts at setting up to get a result!

Repeating multiple times can ramp up costs

Design and receipt of primers can take time especially if re-designs required

This is why we sometimes have long TATs

This is also why we are unable to test multiple primers for one copy number

change…..….it could take up to a year to get a result if we did that!

What can go wrong?

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Case 1

Region of LOH

Homozygous deletion

arr 5q33.1(151,232,711-151,555,856)x0

•Includes first six exons of GLRA1 gene

•Homozygous deletions associated with hyperekplexia

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Cases

qPCR follow up proband and parents:

Father is normal!!??

Proband Mother Father

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Case 1

Region of LOH

Homozygous deletion

Likely maternal segmental UPD

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Case 2

BBS9 Bardet-Biedel Syndrome

From the NGS panel, several patients appeared to have a homozygous

deletion within this gene

qPCR was consistent with this finding

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Case 2

BBS9

However, when qPCR was repeated for an affected sibling and parent, controls

appeared to have deletions!

qPCR repeated 6 times, with different controls - some controls appeared

homozygous, some heterozygous, some normal.

We could not issue a report and recorded the qPCR as inconclusive.

The controls are parents of micorarray probands who are normal via qPCR for the

amplicon tested at the time (they have not been arrayed or sequenced).

What was going on?

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Case 2

BBS9

A SNP issue? No. The primers included SNP regions but all of the BBS9 patients

were SNP free.

Beth did a lot of investigating and found that a mobile insertion element had been

seen in the 1000 genomes pilot project. This is recorded on the DGV (Database of

Genomic Variants).

Worth bearing in mind that some results could be caused by normal population

variation.

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Case 3

Proband Mother Father Control1 Control2

arr 15q11.2(22,770,422-23,214,340)x1 pat

NIPA1 gene – neurosusceptibility syndrome

NGS follow up cases

qPCR provides more evidence on whether or not the NGS result reflects a real

copy number change

We design primers only within exons

We test two sets of primers from within the exon(s) of interest but cannot use

multiple primers to try to work out the exact size of the copy number change

NGS follow up referrals

Sep 2015 – June 2016

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Average TAT 44

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NGS resultconfirmed

NGS result notconfirmed

qPCR notpossible

Inconclusive

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Workflow

Cyto duty scientist receives microarray follow up referral

OR

molecular scientist emails Cyto qPCR team

Senior scientist activates for qPCR on our LIMS

qPCR

Enter result on OMNI

NGS/other follow up:

- internal qPCR OMNI report

Micorarray follow up:

- proband array report updated,

- parental reports issued on

OMNI

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Summary

qPCR is great for detecting specific copy number changes

Relatively cheap

Can be used for copy number changes down to a minimum of 100bp

Freedom to design primers anywhere in the genome

But

Repeats are often necessary so TATs often go over 28 days.

If we have tested a family member previously, it doesn’t necessarily mean

that testing another will be quick!

qPCR can’t tell you the extent of the copy number change

We can’t test multiple primer pairs for one copy number change

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CONCLUSION

We will continue to use it particularly for NGS follow ups as these often

have single exon deletions which are difficult to confirm any other way.

It is considerable cheaper than running an array.

For each patient cost excluding labour is approx. £250 for an array

compared to around £25 for qPCR.

Success rate is also good – we rarely fail cases.

The usual problem is that we run out of DNA after all of the NGS work

and then have to ask for a new sample!

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ANY QUESTIONS ?