Monozygotic twins, also known as monovular twins, share an identical genetic heritage because they are two individuals who derive from the same zygote. For this reason, they have been considered indistinguishable. They represent a limit for the application of markers and analytical methods that are routinely used in forensic science because analyses of DNA fragments (short tandem repeats analysed by capillary electrophoresis) are unable to distinguish monozygotic twins. The recent introduction of ultra-deep next generation sequencing in forensic genetics, also known as massively parallel sequencing, has made it possible to identify a number of genetic variations through genome sequencing (such as copy number variations, single nucleotide polymorphisms and DNA methylation) that make it possible to distinguish monozygotic twins. Here, we present a case of ascertaining biological paternity, in which the alleged father had a monozygotic twin brother. This case led to the examination of international law in similar cases in which the only available biological evidence derives from classical forensic genetic analysis, performed with short tandem repeat (autosomal and/or gonosomal) capillary electrophoresis and the probative value, if recognised, of the next generation sequencing technology in the courtroom.
