# Copy Number Variation: Methods and Clinical Applications
## 2 Methods of CNV Detection
### Methods of Molecular Biology
Their detectability strongly depends on the length and type of the CNV, as well as on its position with regard to the used amplification primers (for more details on the possible effect of CNVs on reliability, see the next subheading). Since 2005, when the first platforms of second-generation sequencing (2 GS) technology became available [38], methods based on MPS have undergone several modifications, their cost ever on the decrease [39]. In current times, 2 GS represents a valuable tool for clinical diagnostics and provides a sensitive and accurate approach for the detection of the major types of genomic variations, including CNVs [40]. There are three main strategies for 2 GS-based CNV analysis, namely whole-genome, whole-exome, and targeted sequencing. Due to the limited length of DNA fragments sequenced by 2 GS sequencers, variation is detected by abnormalities in the affected areas using robust statistical and bioinformatic processing [41]. Read-depth methods highlight regions with an irregular number of sequenced fragments: a loss is seen as a lower, and a gain as a higher than expected amount of a particular segment. Read-pair and split-read approaches analyze fragments with discordant alignments of sequenced fragments, where portions of a single fragment are aligned to unexpected sites in the reference genome. While the above methods directly analyze reads mapped to the reference genome, assembly-based methods compare longer sections of an individual's genome, called contigs. This approach may reveal more complex genome rearrangements, but genome assembly is computationally more intensive and requires substantially higher capacity. Whole-genome sequencing combined with sophisticated computational strategies improved CNV detection, allowing even base-pair resolution of breakpoints [42]. On the other hand, whole-exome sequencing targets only the protein-coding part of the genome. However, since most of the known disease-causing mutations fall into this category, exome sequencing significantly reduces sequencing cost in medical applications and is still sufficiently powerful. Moreover, targeted sequencing provides a greater depth of coverage in regions of interest for an even lower cost [43]. Third-generation sequencing (3 GS) technologies (e.g., single-molecule real-time sequencing [44] and nanopore sequencing [45]) bring promise for better characterization of genomic structural variants due to longer reads [46] that can be more confidently aligned to repetitive sequences, often mediating the formation of structural variants [47]. While both microarray and 2 GS techniques are based on complex laboratory procedures which require several days to obtain results [48], nanopore-based 3 GS provides pocket-sized, low-cost devices that usually take from 24 to 48 h to run, with reads generated continuously, so data can be used for processing and further analysis in real-time during the ongoing sequencingprocess [45]. Moreover, the method can be combined with a rapid library preparation kit capable of obtaining ready to sequence genomic DNA in 10 min. Data generated in the first tens of minutes of a run are sufficient to detect large chromosomal alterations with a resolution in the order of tens of Mb. Data produced in the first 6-12 h of a sequencing run can be used to identify CNVs with an accuracy comparable to currently available array-based methods, and are capable of predicting the allelic fraction of genomic alterations with high accuracy [42]. The problem with CNV breakpoint identification often encountered in PCR- or array-based methods can also be solved by breakpoint sequencing [49]. Using 3 GS devices, it will soon be possible to perform a cost-effective high-resolution molecular karyotyping of the human genome within an hour from sample extraction, allowing ultra-fast analyses in fields where time matters, such as precision oncology and prenatal diagnostics [42].