Next generation sequencing (NGS) has been carried out for a number of purposes in medicine, anthropology, population and forensic genetics. It can produce massive sequencing data, and is expected to become and alternative or an additional approach to overcome the limitation of capillary electrophoresis(CE)- based forensic STR typing, especially in the analyses of mixed samples. However, there has been no available multiplex PCR system that is optimized for NGS analysis of STRs. In this regard, we constructed a multiplex PCR system for NGS analysis STRs, which composed of 4 markers of commonly used commercial kits(vWA, D7S820, D21S11 and D8S1179). Amplicons were designed to have a size ranging about 200 bp which is compatible with the read length of currently available NGS platforms. To validated the multiplex PCR system, PCR products were generated from single-source samples and the mixed DNA samples in a varying ratios, and subsequent barcodeed library was prepared and sequenced on a benchtop sequencer, GS Junior Sequencer(Roche). STR typing results obtained from NGS analysis were consistent with those from CE-based analyses both for single-source samples and mixed samples. As a result, STR types are not easy to distinguish major and minor contributor in mixed DNA samples. In contrast, NGS sequencing was able to differentiate among them by DNA sequence variation. This results suggest that NGS can be one of the good method to indicate who is the contributor in mixed DNA which was originated from crime scene evidence. In addition, we examined DNA sequence of 6 microvariants in D21S11 locus using NGS to find out particular character of D21S21 microvariants. They showed mutations and sequence variations in the repeat region of D21S11 short tandem repeat (STR) loci.
II. 재료 및 방법
1. 실험 시료
2. 실험 방법
1. 표준 DNA의 A-STR형 분석
2. 표준 DNA 9947 A과 9948을 이용한 검출 최소농도의 확인
3. 혼합 DNA에서 혼합비율에 따른 검출여부 확인
4. 사건현장의 증거물 DNA에서 microvariants 검출 및 NGS data 분석