An Investigation into Sequencing Methodologies used to aid in the Differentiation of DNA from Identical Twins

Abstract

Forensic case work uses short tandem repeat profiling for determining the DNA profiles of individuals that may have been linked to a crime scene. However, this method is unable to differentiate between the DNA of monozygotic twins. In these circumstances, it is unclear which twin is responsible. This has been addressed, by other authors, through mutation analysis of the whole genome, however this is an expensive approach and therefore not easily accessible to most forensic labs. The aim of this thesis was to develop and test suitable methodologies to aid in the differentiation of identical twins. As part of this project, two methodologies were explored. Firstly, examining STR sequences for sequence variation in DNA from buccal swabs of four pairs of identical twins. This was done using massively parallel sequencing accompanied by an Ampliseq™ custom primer panel for use on the Ion Torrent PGM™ system. The results of this testing showed that sequence variation among the STRs tested did not highlight sequence differences between the DNA from identical twins. Another approach has been centered in the study of DNA methylation patterns among identical twins, as these patterns change in response to environmental stimuli therefore differing patterns may be found between monozygotic twins. This methodology, focussed on developing and optimising a methodology for the assessment of DNA methylation patterns among identical twins at a specific region of the human genome (IGF2 Shore). Buccal swabs, from five pairs of identical twins underwent the steps of extraction, quantification, amplification, bisulphite conversion and sequencing. Differences in DNA methylation patterns were found among all twin pairs based on differences in methylation patterns at 11 CpG sites. This study has illustrated that the DNA methylation methodology has produced promising results and should be replicated on other body fluids. With careful consideration of the optimisation process, this method can potentially be a viable option for the differentiation of DNA from monozygotic twins.