Carolyn Dunn, Annabel Whibley, Lionel Willatt and Ingrid Simonic Development of SYBR Green RT-qPCR to confirm small SNP array aberrations Carolyn Dunn, Annabel Whibley, Lionel Willatt and Ingrid Simonic Cambridge

Overview of Array Results - 2007 134 SNP Arrays - dev delay - congenital abnormalities 60% Normal Array Result 40% ? Del/dup Array Result Unsuitable for FISH: del <150kb or dup <1.5Mb (18%) FISH confirmatory studies (22%)

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 Fluorescent Probes SYBR Green Low cost

SYBR Green RT-qPCR Principles Denaturation of DNA to produce ssDNA Thermal Cycling: Primers anneal to and extend target sequence SYBR Green I binds to dsDNA and emits a fluorescent signal As PCR amplification proceeds, (causing the amount of dsDNA to increase), the fluorescence signal increases proportionately 3’ ssDNA 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

SYBR Green RT-qPCR Strategy Select target gene in UCSC/Ensembl Export and repeat mask sequence Primer design – Primer3 SNP check (Manchester Diagnostic SNPCheck) and BLAST primer sequences PCR reaction efficiency (90–110%) and precision (Rsq value >0.985) Each primer 15-30bp Tm of two primers within 2ºC of each other GC content 45-55% Try to avoid 3’ terminal T Product size 80-150

Overview of Primer Validations 24 sets of primers 2 taken from RTPrimerDB 22 designed using Primer3 2 Failed QC: reaction efficiency <90 or >110% or Rsq < 0.985 Passed QC 20 Passed QC Re-design Primers

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) dup(5)(p15.3p15.3) dup(12)(q24.32q24.32) dup(5)(p14.3p14.3) Deletion del(5)(p14.3p14.3) Initially some results were not reliable – duplicate reps were replaced with triplicate Reaction volume was reduced to 10ul but some SDs were high so back to 20ul Conflicting result: Duplication by karyotype and FISH but deletion by qPCR! Patient with a deletion of same gene showing deletion by qPCR

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’ A high number of “calls” on the array analysis One of the two array analysis packages highlighted this as an abnormality Summary of data from 2 qPCR experiments

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 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 Summary of data from 2 qPCR experiments

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

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