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Table 1 Common methodologies used for copy number variation detection

From: Appraisal of the technologies and review of the genomic landscape of ductal carcinoma in situ of the breast

Method Brief outline of method Advantages Disadvantages Resolution Main use
Chromosome comparative genomic hybridisation (CGH) Target DNA and normal reference DNA differentially labelled and applied to metaphase spread from cultured normal lymphocytes Genome-wide analysis Cannot detect balanced chromosomal alterations or polyploidy. Resolution limited by use of highly condensed metaphase chromosomes High-level amplification 250 kb Gains 2 Mb Losses 10 to 20 Mb [16] Discovery studies
Array CGH (aCGH) Target DNA hybridised to DNA clones (for example, bacterial artificial chromosomes) or oligonucleotides placed at certain intervals through genome. Genome-wide analysis Cannot detect balanced chromosomal alterations or polyploidy. Prone to spatial bias. Determined by density of clone coverage Discovery studies
Single-nucleotide polymorphism (SNP) arrays Target DNA hybridised to oligonucleotides specific to SNPs and compared with collection of controls Can detect loss of heterozygosity (LOH) and mutations. Normal reference DNA not required. May not be genome-wide analysis as SNPs are unevenly distributed across genome; however, commercially available arrays deliberately include probes in SNP-poor areas to increase genome coverage. Prone to spatial bias. Determined by length, density, and distribution of probes Discovery studies
Molecular inversion probe array Target DNA amplified in SNP-dependent manner and hybridised to oligonucleotides Suitable for small amounts (<100 ng) of degraded DNA. Can detect LOH and mutations. As for SNP arrays Determined by density and distribution of probes Discovery studies
Massively parallel sequencing Parallel sequencing of large numbers (potentially millions) of templates Potential genome-wide analysis. Can identify copy number neutral structural variations. Suitable for fragmented DNA. Large volume of sequencing and data analysis Potential single-base resolution Discovery studies
Fluorescence in situ hybridisation Fluorescently labelled genomic clones hybridised to target interphase nuclei Structural rearrangements and polyploidy can be detected. Minimal multiplexing ability 50 kb [17] Locus-specific copy number analysis
Quantitative polymerase chain reaction (PCR) Quantitation of copy number based on rate of amplification Low DNA input requirements Limited multiplexing ability. Prone to PCR amplification bias. Precision dependent on number of replicates. Underestimates high copy numbers. Assay design dependent, but resolution of less than 100 base pairs (bp) possible. Locus-specific copy number analysis
Droplet digital PCR Quantification of copy number based on Poisson distribution statistics of thousands of digital PCRs [18] Low DNA input requirements and compatible with fragmented DNA Minimal multiplexing ability. Cannot detect polyploidy. Targets regions of less than 100 bp possible. Can detect more than 0.15 % positive droplets per sample [19]. Locus-specific copy number analysis
Multiplex amplification and probe hybridisation (MAPH)/multiplex ligation-dependent probe amplification Quantification of PCR products of hybridised probes Multiplexable Large amount of good-quality DNA required for MAPH (250 to 1,000 ng, >100 bp) [20] 150 bp [21, 22] Locus-specific copy number analysis
Nanostring nCounter system Absolute quantification of probes hybridised to target region Multiplexable. Requires fragments of 100 bp or greater Requires 300 ng of input DNA Detects 0 to 4 copies of minimum 100 bp target regions Locus-specific copy number analysis