차세대 염기서열 분석법을 이용한 15개 상염색체 STR의 염기서열 생성 및 유전자형 분석
Sequence Generation and Genotyping of 15 Autosomal STR Markers Using Next Generation Sequencing
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Recently, next generation sequencing (NGS) has received attention as the ultimategenotyping method to overcome the limitations of capillary electrophoresis (CE)-based short tandem repeat (STR) analysis, such as the limited number of STR locithat can be measured simultaneously using fluorescent-labeled primers and the maximumsize of STR amplicons. In this study, we analyzed 15 autosomal STR markersvia the NGS method and evaluated their effectiveness in STR analysis. Using maleand female standard DNA as single-sources and their 1:1 mixture, we sequentiallygenerated sample amplicons by the multiplex polymerase chain reaction (PCR)method, constructed DNA libraries by ligation of adapters with a multiplex identifier(MID), and sequenced DNA using the Roche GS Junior Platform. Sequencing datafor each sample were analyzed via alignment with pre-built reference sequences. Most STR alleles could be determined by applying a coverage threshold of 20% forthe two single-sources and 10% for the 1:1 mixture. The structure of the STR in eachallele was accurately determined by examining the sequences of the target STRregion. The mixture ratio of the mixed sample was estimated by analyzing the coverageratios between assigned alleles at each locus and the reference/variant ratiosfrom the observed sequence variations. In conclusion, the experimental method usedin this study allowed the successful generation of NGS data. In addition, the NGS dataanalysis protocol enables accurate STR allele call and repeat structure determinationat each locus. Therefore, this approach using the NGS system will be helpful to interpretand analysis the STR profiles from singe-source and even mixed samples inforensic investigation.
Abstract
Recently, next generation sequencing (NGS) has received attention as the ultimategenotyping method to overcome the limitations of capillary electrophoresis (CE)-based short tandem repeat (STR) analysis, such as the limited number of STR locithat can be measured simultaneously using fluorescent-labeled primers and the maximumsize of STR amplicons. In this study, we analyzed 15 autosomal STR markersvia the NGS method and evaluated their effectiveness in STR analysis. Using maleand female standard DNA as single-sources and their 1:1 mixture, we sequentiallygenerated sample amplicons by the multiplex polymerase chain reaction (PCR)method, constructed DNA libraries by ligation of adapters with a multiplex identifier(MID), and sequenced DNA using the Roche GS Junior Platform. Sequencing datafor each sample were analyzed via alignment with pre-built reference sequences. Most STR alleles could be determined by applying a coverage threshold of 20% forthe two single-sources and 10% for the 1:1 mixture. The structure of the STR in eachallele was accurately determined by examining the sequences of the target STRregion. The mixture ratio of the mixed sample was estimated by analyzing the coverageratios between assigned alleles at each locus and the reference/variant ratiosfrom the observed sequence variations. In conclusion, the experimental method usedin this study allowed the successful generation of NGS data. In addition, the NGS dataanalysis protocol enables accurate STR allele call and repeat structure determinationat each locus. Therefore, this approach using the NGS system will be helpful to interpretand analysis the STR profiles from singe-source and even mixed samples inforensic investigation.
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