Development of SYBR Green RT-qPCR to confirm small SNP array aberrations

Carolyn DunnCarolyn Dunn, Annabel Whibley, Lionel , Annabel Whibley, Lionel Willatt and Ingrid SimonicWillatt and Ingrid Simonic

Cambridge

Overview of Array Results - 2007

134 SNP Arrays - dev delay

- congenital abnormalities

Unsuitable for FISH: del <150kb

or dup <1.5Mb (18%)

60% Normal Array Result

FISH confirmatory

studies (22%)

40% ? Del/dup Array Result

Options for Confirmatory Studies

• A second type of array– Re-analysis of whole genome– High set-up and running costs

• MLPA– Able to multiplex– Cost of probe expensive for single family follow-up

studies• RT-qPCR

I. Fluorescent ProbesII. SYBR Green

• Low cost

SYBR Green RT-qPCR

Principles1. Denaturation of DNA to produce

ssDNA2. Thermal Cycling:

• Primers anneal to and extend target sequence

3. SYBR Green I binds to dsDNA and emits a fluorescent signal

4. As PCR amplification proceeds, (causing the amount of dsDNA to increase), the fluorescence signal increases proportionately

3’ 5’

3’ 5’

N.B. SYBR Green I binds all dsDNA (including primer-dimers and non-specific reaction products) – essential that primers are specific to target sequence

ssDNA

SYBR Green RT-qPCR

Strategy1. Select target gene in UCSC/Ensembl 2. Export and repeat mask sequence 3. Primer design – Primer3 4. SNP check (Manchester Diagnostic

SNPCheck) and BLAST primer sequences5. PCR reaction efficiency (90–110%) and

precision (Rsq value >0.985)

Overview of Primer Validations

24 sets of primers

2 taken from RTPrimerDB

22 designed using Primer3

Re-design Primers

Passed QC 20 Passed QC2 Failed QC: reaction efficiency <90 or >110% or Rsq < 0.985

Proof-of-principle Study

• Is this approach reliable and robust to use diagnostically?

• Which real time PCR machine to use? ABI 7900 versus Rotor-Gene 65H0– Ease of use, cost, consumables– Plates versus tubes on a rotor

• 6 cases (5 duplications and 1 deletion)– Abnormal karyotype (4) or array result (2)– Confirmed by FISH

Set-up and Analysis

• 4 controls• GAPDH used as the reference gene• All reactions in triplicate - SD <0.18• Each experiment replicated• Analysed using ∆∆Ct method and primer efficiency-

corrected

• Expected relative copy number – Normal: 1.0 (0.85-1.15)– Deletion: 0.5 (0.35-0.65)– Duplication: 1.5 (1.35-1.65)– Equivocal: 0.65-0.85 and 1.15-1.35

Proof-of-principle Study Results

Abnormality qPCR confirmation

dup(2)(q14.2q14.2) Yes

dup(7)(q11.23q11.23) Yes

dup(5)(p15.3p15.3) Yes

dup(12)(q24.32q24.32) Yes

dup(5)(p14.3p14.3) Deletion

del(5)(p14.3p14.3) Yes

SNP Array Follow-up Data (I)

• 6 SNP array abnormalities followed-up by qPCR to date (5 patients)

– 1 was not confirmed – within the ‘normal range’

?del 14q21.3

0.981.09

0.00

0.50

1.00

1.50

Control average Patient average

Sample

Re

lative

CN

Summary of data from 2 qPCR experiments

• A high number of “calls” on the array analysis

• One of the two array analysis packages highlighted this as an abnormality

SNP Array Follow-up Data (II)

• 6 SNP array abnormalities followed up to date (5 patients)– 1 was not confirmed - same CN as controls– 1 borderline equivocal/duplication result

• primer pair failed QC• Re-design primers and repeat

?dup12q21.1-q21.2

1.02

1.28

0.00

0.50

1.00

1.50

Control average Patient average

Sample

Re

lati

ve

CN

Summary of data from 2 qPCR experiments

SNP Array Follow-up Data (III)

• 6 SNP array abnormalities followed up to date (5 patients)

– 1 was not confirmed - same CN as controls– 1 borderline equivocal/duplication result

• primer pair failed QC• repeat with second set of primers

– 4 confirmed (2 dels and 2 dups)

SNP Array Follow-up Data (IV)

• 300kb deletion (no suitable FISH clone)

• 110kb deletion

• 42kb duplication

?del7q11.21

0.99

0.39

0.00

0.50

1.00

1.50

Control average Patient average

Sample

Re

lativ

e C

N

?dup2q37.3

1.07

1.58

0.00

0.50

1.00

1.50

2.00

Control average Patient average

Sample

Re

lativ

e C

N

?del9q33.1

1.07

0.37

0.00

0.50

1.00

1.50

Control average Patient average

Sample

Re

lativ

e C

N

Summary of data from 2 qPCR experiments

SNP Array Follow-up Data (VI)• 660kb ?dupXq27.1 (includes SOX3 gene)

0

1

2

3

4

5

6

7

8

Female C (n=5) Male C (n=5) Patient X

Sample

Re

lativ

e C

op

y N

um

be

r

X ?dupX0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

Female C(n=9)

Male C(n=8)

XXXXY Patient X

Sample

Rel

ativ

e C

opy

Num

ber

SOX3

Summary

• Costs higher than first predicted as primer re-design required for some cases

• Equivocal result• Primers that fail QC step

• A copy number of 4 or greater may not be accurately detected

• A promising option for verifying small array deletions or duplications

Acknowledgments

• Dr Lucy Raymond (Clinical Genetics,

Addenbrooke’s Hospital) • Dr Martin Curran (Head of Molecular Diagnostic

Microbiology Section, Addenbrooke’s Hospital)