Identification of the perpetrator among identical twins using next-generation sequencing technology: A case report

Background: Monozygotic (MZ) twins, considered genetically identical, cannot be distinguished using regular short tandem repeats (STR) typing, thus presenting a challenge for forensic geneticists. In paternity testing, single nucleotide polymorphisms (SNPs) in nuclear DNA can help distinguish MZ twins. However, the unique features of the mitochondrial genome, such as high copy number, small genome size, and high substitution rate, make it a promising source for applications in forensic science. Methods: Whole-genome sequencing (WGS) was performed on blood samples, and bioinformatic analysis was used to distinguish between MZ twins. Amplification refractory mutation system polymerase chain reaction (ARMS-PCR) was used to confirm the WGS results. This methodology was further applied to forensic samples from criminal cases. Amplicon sequencing was also performed to further exclude the innocent twin. Results: The monozygosity of the twins was confirmed using STR typing. Only one potential somatic mutation, m.6903 T > C (2.6%), in the mitochondrial genome of one of the twins was verified when the sequence depth was set to 2000-fold, while no other distinguishing locus in the nuclear genome was identified. By dividing the number of C-reads by total reads, WGS data confirmed the amount of the minor component C to be 2.6%, which was further confirmed by ARMS-PCR. In addition, the heterogeneous locus was used to identify samples obtained from four criminal cases for forensic testing. Two heterogeneous loci in the sperm DNA of the other twin were identified by amplicon sequencing, and the amount of minor component T in m.6935C > T and m.6938C > T was estimated to be 17.91% and 18.79%, respectively. Conclusion: The biological samples taken from the MZ twins were distinguished using a combination of WGS, allele-specific PCR, and deep-amplicon sequencing. Compared with nuclear DNA, mitochondrial DNA exhibited a higher potential for distinguishing between the MZ twins. The distinguishing feature of the mitochondrial DNA was the heterogeneous SNPs that occurred in only one twin. One SNP was further verified in the samples from the criminal cases and helped identify the perpetrator in case 1 and case 2. Furthermore, two heterogeneous SNPs found by amplicon sequencing helped to exclude the innocent twin in all four cases. Our findings demonstrated that a combination of deep sequencing and molecular analysis can be an effective way to distinguish between identical twins and can be used to analyze samples from criminal cases.