Korean J Leg Med.  2014 May;38(2):48-58. 10.7580/kjlm.2014.38.2.48.

Sequence Generation and Genotyping of 15 Autosomal STR Markers Using Next Generation Sequencing

• Kim EH
• Jung SE
• Shin KJ
• Yang WI
• Yang IS

• KMID: 1849353
• DOI: http://doi.org/10.7580/kjlm.2014.38.2.48

Abstract

Recently, next generation sequencing (NGS) has received attention as the ultimate genotyping method to overcome the limitations of capillary electrophoresis (CE)-based short tandem repeat (STR) analysis, such as the limited number of STR loci that can be measured simultaneously using fluorescent-labeled primers and the maximum size of STR amplicons. In this study, we analyzed 15 autosomal STR markers via the NGS method and evaluated their effectiveness in STR analysis. Using male and female standard DNA as single-sources and their 1:1 mixture, we sequentially generated 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 data for each sample were analyzed via alignment with pre-built reference sequences. Most STR alleles could be determined by applying a coverage threshold of 20% for the two single-sources and 10% for the 1:1 mixture. The structure of the STR in each allele was accurately determined by examining the sequences of the target STR region. The mixture ratio of the mixed sample was estimated by analyzing the coverage ratios between assigned alleles at each locus and the reference/variant ratios from the observed sequence variations. In conclusion, the experimental method used in this study allowed the successful generation of NGS data. In addition, the NGS data analysis protocol enables accurate STR allele call and repeat structure determination at each locus. Therefore, this approach using the NGS system will be helpful to interpret and analysis the STR profiles from singe-source and even mixed samples in forensic investigation.

Keyword

Short tandem repeat; Next generation sequencing; Repeat structure; Sequence variation; Mixture

MeSH Terms

Alleles
DNA
Electrophoresis, Capillary
Female
Gene Library
Humans
Ligation
Male
Microsatellite Repeats
Multiplex Polymerase Chain Reaction
Statistics as Topic
DNA

Figure

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  Fig. 1. Schematic view of STR reference sequences. Long flanking sequences ranged between 500 bp and 550 bp in STR reference sequences were designed for complete alignment of sample sequences that generated with any primer combinations.

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  Fig. 2. Quality check of constructed libraries on High Sensitivity chip using 2100 Bioanalyzer. Fragments less than 100 bp including adaptor dimers were successfully removed. a: Standard male DNA 2800M; b: Standard female DNA 9947A; c: 1:1 mixture

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  Fig. 3. Estimation of mixture ratio based on reference/variant ratios from observed sequence variations in D13S317 locus. The sequence variation of adenine (A) to thymine (T) was detected in 3´ flanking region of D13S317 locus. Mixture ratio was estimated to 46% (A) : 53% (T). a: Standard male DNA 2800M; b: Standard female DNA 9947A; c: 1:1 mixture; d: Mixture ratio

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