Table 6.

Comparison of selected diagnostics methods.

Method	Advantages, Applications	Disadvantages, Limitations
Next-generation sequencing (NGS)	sensitivity and specificity >99% [77], massively parallel sequencing in several genes simultaneously, a relatively short time of analysis, detection of low input of DNA samples [86,88], detection of SNVs and small insertions/deletions [94,95]	advanced bioinformatics systems and large data storage potential [96,97], filtering and data interpretation (various variants can be found when a large number or whole genes are sequenced) [94,97], issues with detecting structural rearrangements or copy number variations (CNVs) [95]
Multiplex Ligation-dependent Probe Amplification (MLPA)	wide diagnostic applications—copy numbers, point mutations detection, methylation profiling, also detected simultaneously, washing unbounded probes are not necessary, a simple and cost-effective method, easy analysis of the results [91,92]	does not detect balanced mutations, like balanced translocations or inversions (detects only ones which affect the probe binding sequence), probes can be designed only for known mutations—impossible to detect an unknown mutation, the heterozygous deletions analysis is reliable when tumor cells constitute 20–30% of the sample, heterozygous duplication—about 40% [91,92], does not provide precise deletion/insertion characteristics‘ [98]
High-Resolution Melting (HRM)	simple after proper optimization, fast, high-throughput, software supporting optimization available, relatively simple and not-expensive equipment needed [98,99]	detected variants not characterized, further characterization with another method, e.g., sequencing needed [98,99]
Sanger sequencing	the gold standard, mainly for detecting point mutations, high quality reads [98]	not cost-effective when a large number of samples and long sequences are analyzed, technically demanding method [98]
Single-Strand Conformation Polymorphism (SSCP)	detection of point mutations, deletions, and insertions, detection of unknown variants, simple and quite fast method [81]	low sensitivity and repeatability, amplicons not longer than 200–300 bp, detected variants not characterized, further characterization with another method, e.g., sequencing needed [80]
Conformation-sensitive Gel Electrophoresis (CSGE)	detection of single-nucleotide mutations, small insertions, and deletions, relatively high sensitivity and specificity, cost-effective [84]	detected aberrations need to be sequenced time-consuming method [84]
Denaturing Gradient Gel Electrophoresis (DGGE)	detection of unknown variants [80], relatively cheap, reliable heteroduplexes detection [98]	technically demanding, results must be characterized by another method, e.g., sequencing [80,98], GC-rich regions can be difficult to optimize and analyze [98,100]
Denaturing High-Pressure Liquid Chromatography (DHPLC)	sensitivity nearly 100% [82], a wide spectrum of applications: mutations and SNP detection, gene mapping, gene expression and methylation analysis [82,101], does not require modified primers or specific reagents [101], relatively cheap [81]	does not detect copy number aberrations [92], detected variants need to be characterized by sequencing, when more than one melting domain in tested amplicon-analysis of several temperatures required [82], chemical waste generation, not a high-throughput method [85]
Southern blot	detection of large insertions/deletions [82]	not always small deletions are detected [82] time-consuming [98]