Introduction
Copy number changes in ductal carcinoma in situ
Detection methods
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] | 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 |