Deep-sequencing technologies and potential applications in forensic DNA testing

Development of second- and third-generation DNA sequencing technologies have enabled an increasing number of applications in different areas such as molecular diagnostics, gene therapy, monitoring food and pharmaceutical products, biosecurity, and forensics. These technologies are based on different biochemical principles such as monitoring released pyrophosphate upon incorporation of a base (pyrosequencing), fluorescence detection subsequent to reversible incorporation of a fluorescently labeled terminator base, ligation-based approach wherein fluorescence of a cleaved nucleotide after ligation is measured, measuring the proton released after incorporation of a base (semiconductor-based sequencing), monitoring incorporation of a nucleotide by measuring the fluorescence of the fluorophore attached to the phosphate chain of the nucleotide, and by detecting the altered charge in a protein nanopore due to released nucleotide by exonuclease cleavage of a DNA strand. Analysis of multiple DNA fragments in parallel increases the depth of coverage while decreasing labor, cost, and time, highlighting some major advantages of deep-sequencing technologies. DNA sequencing has been routinely used in the forensic laboratories for mitochondrial DNA analysis. Fragment analysis, however, is the preferred method for short tandem repeat genotyping due to the cumbersome and costly nature of first-generation DNA sequencing methodologies. Deep-sequencing technologies have brought a new perspective to forensic DNA analysis. Studies include short tandem repeat (STR) analysis to reveal hidden variation in the repeat regions, mtDNA sequencing, single nucleotide polymorphism (SNP) analysis, mixture resolution, and body fluid identification. Recent publications reveal that attempts are being made to expand the capability.