Table 1.

Laboratory techniques for CNVs detection.

Chromosome banding: Chromosomes are prepared from dividing cells, stained, and viewed with a microscope. Large deletions, duplications, and translocations are detected if the banding pattern or chromosome structure is altered. However, smaller microdeletions and microduplications are not observed

FISH: The technique consist in fluorescent-labeled DNA probes that hybridize to metaphase or interphase cells to visualize a locus on a chromosome and determine copy number. FISH can determine the location of chromosomal segments identified by microarray, NGS, and WGS

qPCR: This is considered a high throughput technique for identifying CNVs. The cycle number (Ct) is proportional to the amount of starting template so the Ct values of the target gene can be compared to unrelated reference sequences that do not differ in copy content, so generating data which are used for CNVs analysis

MLPA: This method is able to analyze in a single reaction up to 50 DNA sequences and to detect copy number variation of specific genes, including small intragenic rearrangements. It has been utilised in recent years for the detection of CNVs and/or the validation of array-CGH results

Microarrays: Array-CGH detects copy-number differences between abnormal and reference genomes, but lack in the identification of balanced chrosmosomal rearrangements. SNP-array detect changes in copy-number and allelic ratios. Microarray analysis is not the best method to determine CNVs location and SV organization

WGS: Sequencing the whole genome provides the most comprehensive SV analysis. Breakpoints of CNV and copy-neutral SV are detectable by paired-end reads that have discordant mappings to the reference genome. Complex genomic structures identified by WGS may require FISH or chromosome banding to place rearranged segments

CGH = Comparative Genomic Hybridization; CNVs = Copy Number Variants; FISH = Fluorescence In Situ Hybridization; qPCR = Quantitative PCR; SNP = Single Nucleotide Polymorphisms; SV = Structural Variants; WGS = Whole Genome Sequencing.