Accurate and objective copy number profiling using real ...

1 Review ArticleAccurate and objective copy number profiling using real-time quantitative PCRB arbara D haenea, Jo Vandesompelea,b, Jan Hellemansa,b,*aCenter for Medical Genetics, Ghent University Hospital, Ghent, BelgiumbBiogazelle, Ghent, Belgiumarticle infoArticle history:Accepted 14 December 2009 Available online 6 January 2010 Keywords:qPCRCNVCopy number variationsValidationExperiment designQuality controlabstractCopy number changes are known to be involved in numerous human genetic disorders. In this context,qPCR-based copy number screening may serve as the method of choice for targeted screening of the rel-evant disease genes and their surrounding regulatory landscapes. qPCR has many advantages over alter-native methods, such as its low consumable and instrumentation costs, fast turnaround and assaydevelopment time, high sensitivity and open format (independent of a single supplier).

2 In this chapterwe provide all relevant information for a successfully implement of qPCR-based copy number emphasize the significance of thoroughin silicoand empirical validation of the primers, the needfor a well thought-out experiment design, and the importance of quality controls along the entire work-flow. Furthermore, we suggest an appropriate and practical way to calculate copy numbers and to objec-tively interpret the provided guidelines will most certainly improve the quality and reliability of your qPCR-basedcopy number screening. 2010 Elsevier Inc. All rights IntroductionCopy number changes under the form of deletions and duplica-tions are known to be involved in numerous human genetic disor-ders. Moreover, each individual s genome embodies several copynumber polymorphisms of various sizes which are thought to con-tribute to normal phenotypic variation and susceptibility to multi-factorial disease[1,2].

3 Hence, it is not surprising that a widespectrum of laboratory methods has been developed to identifythese copy number changes. Well known and widely applied tech-niques include conventional karyotyping, fluorescent in situhybridization (FISH) analysis, microarray-based copy numberscreening, multiplex ligation-dependent probe amplification(MLPA), and quantitative PCR (qPCR)[3,4]. Each method is charac-terized by particular (dis)advantages and the choice for a giventechnique largely depends on the application, required resolution,flexibility, workload, and cost. Conventional karyotyping allowsthe detection of structural variations across the entire genome,but it is limited in resolution (>5 10 Mb). FISH analysis for tar-geted regions has been used in a routine setting for many years,and requires either metaphase chromosomes (similar to karyotyp-ing) or interphase nuclei (resolution approximately 100 kb).

4 Micro-array-based copy number profiling has improved the resolution inthe last decade and facilitated the detection of much smaller copynumber changes[4]. The most recent high density targeted arrayseven achieve a resolution of a few base pairs. For patients withmental retardation or other complex phenotypes, genome widecopy number profiling using microarrays proves to be the mostsuitable approach to reveal the underlying molecular defect[5].In contrast to the research driven race for ever increasing resolu-tion, the majority of diagnostic tests for genetic disorders are re-stricted to the targeted screening of the relevant disease genesand their surrounding regulatory landscapes. In the latter context,focused copy number screening methods are preferentially used,such as MLPA, targeted microarrays, and real-time qPCR, the PCR product accumulation is mea-sured in real-time resulting in a sigmoidal amplification curve.

5 Sev-eral detection chemistries are available to measure productaccumulation, including hydrolysis probes, molecular beacons,dual hybridization probes and double stranded DNA specific bind-ing dyes. There is a relationship between the moment that the fluo-rescent PCR signal increases above the background and the initialamount of input DNA; larger amounts of input material will resultin lower quantification cycle (Cq) values. The Cq value representsthe fractional PCR cycle that is characteristic for the amplificationcurve ( where increase in fluorescence is maximum) or at whichthe fluorescence crosses a certain threshold. qPCR has many advan-tages over alternative methods, such as its low consumable andinstrumentation costs, fast turnaround and assay developmenttime, high sensitivity and open format (independent of a singlesupplier).

6 To date, qPCR is the golden standard for gene expressionanalysis. For copy number determination, qPCR has been less1046-2023/$ - see front matter 2010 Elsevier Inc. All rights *Corresponding author. Address: Center for Medical Genetics, Ghent UniversityHospital, De Pintelaan 185, B-9000 Ghent, Belgium. Fax: +32 9 Hellemans).Methods 50 (2010) 262 270 Contents lists available atScienceDirectMethodsjournal homepage: used, but recent developments hold the promise of tak-ing this application to the next this chapter we provide all relevant information required touse qPCR for copy number Description of the Selection of regionsThe overall accuracy of an answer to a specific research or diag-nostic question largely depends on the number of qPCR assays,their position relative to the disease locus, and the spatial intervalbetween subsequent amplicons.

7 First, the genes or intergenic re-gions of interest are selected. The number of selected genes usuallyaffects the number of assays included per gene. When a large seriesof genes needs to be screened, the number of assays per gene is of-ten restricted to one or two due to practical and financial con-straints. If a copy number variant (CNV) in a given gene hasalready been associated to a specific disorder or phenotype, thensuch a gene may need to be screened in much more detail to en-sure the detection of small deletions. Ultimately, one often wantsto screen for CNVs in every single exon, taking into account the dif-ferentially spliced exons. The latter would require the developmentof at least one assay for each exon. Increasing the number of ampli-cons will increase both the resolution and the cost per , there is a tradeoff between cost per sample and the CNVdetection ratio.

8 In conclusion, the number of amplicons and thespacing between successive amplicons largely depends on the spe-cific research or diagnostic question. In addition, it may be impor-tant to consider the screening cost per Assay design and Primer design and in silico quality controlDedicated design of the qPCR primers and extensivein silicoandempirical validation are the key for proper selection of the region to be quantified is obviouslythe starting point for primer design. The DNA sequence for a geneor for a genomic region can easily be retrieved from the UCSC gen-ome browser ( ). The genomic position of aregion or the name of a gene can be entered in the Human GenomeBrowser Gateway . In case of a gene name, the browser allows youto select a specific RefSeq sequence.

9 In the actual Genome Browserwindow you can customize the annotation tracks that are of inter-est, in this case the RefSeq Genes and the latest SNP track. Next,the DNA sequence can be retrieved by clicking on the DNA link onthe top blue menu bar in the Genome Browser. This tool allowscustom configuration of the DNA display, which can be useful tohighlight exonic regions or mask repeats. Protein domains or othersequences that can be expected to have similarities with othergenomic regions are to be avoided to reduce the chance of nonspe-cific pairs for the region of interest should be designed accord-ing to stringent parameters to ensure successful assays and conve-nient experiment design. Practical primer design tools for thisapplication are the PrimerQuest software (Integrated DNA Technol-ogies, ) and Primer3 Plus ( ).

10 Primer design parameters: Primerslength:9 bp/20 bp/30 bp(minimum/optimal/maximum). Melting temperature (Tm) primers: 58 C/59 C/60 C (mini-mum/optimal/maximum). MaximumTmdifference between primer pairs: 2 C. GCcontentprimers:30%/50%/80%(minimum/opt imal/maximum). Amplicon length: 80 150 bp. The five nucleotides at the 30-end should have no more than twoG or C bases. This can be set by adjusting the max 30stabilityparameter. Practice has shown that the optimal maximum 30stability valueis 3, but higher values may generate acceptable primers as well. Avoid runs of four or more identical nucleotides (especially Gbases).Subsequently, the generated primers should be subjected toinsilicovalidation to avoid secondary structures, single nucleotidepolymorphisms (SNPs) and copy number polymorphisms at theannealing sites[6,7].