Table 1 Common methodologies used for copy number variation detection

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