DNA Sequencing
Next generation DNA sequencing (NGS) may soon have a role in relationship and forensic testing. Massively parallel DNA sequencing can reveal the nucleotide sequences of thousands of individual template DNA molecules within a given sample simultaneously. The variation that characterizes an individual sample is far beyond what is revealed through traditional Sanger sequencing, whether the sample is from a single source sample or a sample made up of DNAs from various individuals, such as might be found in an evidentiary sample. NGS is not only used to characterize nuclear DNA and RNA but also has applications for mtDNA analysis that focuses on the entire mitochondrial genome rather than just specific hypervariable regions (HVI and II). In addition, since the template size suited for NGS ranges from 200–400 basepairs, NGS is well suited to provide DNA typing information with badly degraded genomic DNA such as might be recovered from human remains.
In rare cases where monozygotic twins are tested as the alleged father or suspects in a forensic case, it has been demonstrated that NGS can be used to determine single nucleotide differences that can distinguish between ‘identical’ twins (Karow, 2013). Mutations that distinguish the twins occur just before or after the morula splits. In order to be transmitted to a child, they need to be present in the father’s germline. Samples used for the testing has included semen, blood, and buccal swabs; slight differences in the number of informative SNPs occurs in the various tissues, possibly due to mutations that continue to arise during the development of an embryo, with some present in the germline but not in other tissues and vice versa. Samples from both twins must be tested so that the SNPs differing in the twins can be compared to evidence collected at the crime scene. Because of the high error rate of NGS, confirmation of the difference(s) must be confirmed with traditional Sanger sequencing. It is expected that there would be no issues with introducing NGS in court because Sanger sequencing, a well-established method, is used to confirm the findings.
Because of the high number of missing persons cases and mass disasters, NGS is also finding use when mtDNA testing is used. Whole genome sequencing that increases the discrimination power of traditional mtDNA testing is required to identify individuals. It is also a higher throughput method than traditional Sanger sequencing and provides the opportunity to deconvolute mixtures when material contains DNA from more than one individual. It is expected that NGS will replace Sanger sequencing for all but a few forensic mtDNA cases in the next 5 years (Melton, 2014).
A number of NGS kits have recently been developed that can simultaneously amplify autosomal, mitochondrial, and Y-STRs in combination with single nucleotide repeats (SNPs). This substantially increases the amount of data that can be obtained from a single sample but still allows compatibility with established criminal databases such as Combined DNA Index System (CODIS). Although traditional STR typing using capillary electrophoresis is the more cost-effective method, NGS can extend the capabilities of a laboratory to obtain DNA profiles from compromised samples and can provide additional information, such as probable ancestry and physical features of the individual whose DNA is being tested. These kits or similar ones could also be used in relationship testing laboratories, particularly when the sample is a nontraditional one (i.e., not blood or a buccal swab). There are laboratories that are currently using a noninvastive prenatal paternity test (Ryan et al., 2013). Fetal cell-free DNA (cfDNA) typically comprises <20% of the total cfDNA in maternal plasma; cfDNA is highly fragmented. SNP microarrays or NGS has been used to characterize the fetal cfDNA and compare it to the DNA from an alleged father and a mother in a prenatal parentage test. Aggregating data from over 300 000 SNPs allows for highly accurate paternity determinations. The ability to separate out the fetal cfDNA can also allow for testing for chromosomal abnormalities using NGS (Avent, 2012).