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 Table of Contents  
Year : 2018  |  Volume : 4  |  Issue : 2  |  Page : 67-69

The application of next-generation sequencing to validate D12S391 microvariation

1 Department of Forensic Biology DNA, Forensic Science Service of the Beijing Public Security Bureau, Beijing, China
2 Department of Forensic Biology, Beijing Police College, Beijing, China
3 The Jinci College of Shanxi Medical University, Taiyuan, China
4 Department of DNA Testing, Beijing Tongda Shoucheng Institute of Forensic Science, Beijing, China
5 CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China

Date of Web Publication29-Jun-2018

Correspondence Address:
Dr. Qingxia Zhang
Forensic Science Service of the Beijing Public Security Bureau, Beijing 100192
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jfsm.jfsm_62_17

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Objective: In a paternity case, the D12S391 locus was reported as a mismatch. To confirm the existence of mutations and mutations come from father or mother. Methods: STR and next-generation sequencing technology were used to validate the sequence. Results: NGS showed the loss of one adenine between the 19.3 allele of the child and allele 20 of the mother. Conclusion: The NGS can be applied in the paternity to validate the mutation.

Keywords: Mutation, next-generation sequencing, STR

How to cite this article:
Zhang Q, Liu J, Ren H, Sun S, Zhao Y, Chen C, Jia L, Liu Y, Yan J. The application of next-generation sequencing to validate D12S391 microvariation. J Forensic Sci Med 2018;4:67-9

How to cite this URL:
Zhang Q, Liu J, Ren H, Sun S, Zhao Y, Chen C, Jia L, Liu Y, Yan J. The application of next-generation sequencing to validate D12S391 microvariation. J Forensic Sci Med [serial online] 2018 [cited 2022 Nov 26];4:67-9. Available from: https://www.jfsmonline.com/text.asp?2018/4/2/67/235445

  Introduction Top

Short tandem repeat (STR) analysis is widely used in paternity testing. In general, the results of paternity testing conform to Mendelian inheritance, tracing the presence of mutations. Most reported microsatellite mutations are confined to single-step mutations, involving the insertion or deletion of one core repeat. Rarely, microsatellite mutations are multi-step mutations, involving the insertion or deletion of two or more core repeats.[1] In this study, we used next-generation sequencing (NGS) technology to validate STR sequencing in a case of disputed paternity.

  Materials and Methods Top


Blood samples were collected from the mother, the child, and the alleged father. Genomic DNA was extracted using magnetic beads. Informed written consent was attained from all the three participants for the use of the samples for DNA profiling and subsequent research. All experiments of this study were carried out in accordance with the guidelines and regulations of the Ethical committee of our institution and the date of the approval was March 16, 2017.

Capillary electrophoresis short tandem repeat profiling

We used a 28-cycle polymerase chain reaction (PCR) on a PowerPlex 21 system (Promega Corporation, Medicine, USA) and a 27-cycle PCR on a Microreader™ 23sp ID System (Microread Corporation, Suzhou, China) to amplify 38 autosomal STR loci according to the manufacturers' instructions. All amplification reactions were performed with a PE 9700 Thermal Cycler (Perkin Elmer, Norwalk, CT). PCR products were sequenced on an ABI 3130xl Genetic Analyzer and analyzed using GeneMapper ID v3.2 software (Applied Biosystems, Woolston Warrington, UK) for automated profiling. The parentage index was calculated according to the formula provided in the Regulation for parentage testing laboratories of forensic (GA/T 965–2011), using data from the Han population in China.[2]

Next-generation sequencing profiling

The three samples and two 9947A controls were sequenced in one Personal Genome Machine (PGM) Ion 316 V2 BC chip. Using a designed fusion primer pool and DNA polymerase (SeqTypeR ® 24, IPE Biotechnology, Beijing, China), the libraries contained 24 STR amplicons (Amelo, D3S1358, D13S317, D7S820, D16S539, D8S1179, D21S11, D18S51, VWA, D5S818, FGA, TPOX, TH01, D2S1338, CSF1PO, D19S433, D12S391, D17S974, D1S1656, D1S1679, D6S1043, D6S474, PentaD, and PentaE) attached by adapters and codes, and were amplified directly. Fusion primers were designed using the Ion Amplicon Library Preparation method to enable one-directional sequencing.

Each 10 μL PCR reaction contained 2 U DNA polymerase, 1 × PCR buffer, 0.2 μL 10 mM dNTPs, 1 μL primer pool (0.03–0.1 μM of each primer pair), and 1 μL DNA template. The PCR cycling conditions were as follows: 95°C for 5 min, 22 cycles of 95°C for 30 s, 60°C for 30 s, and 68°C for 1 min, and a final extension at 68°C for 10 min.

Coded libraries of different samples were pooled at equal volume (5 μl of each coded library), and the pooled libraries were purified using 1.2 volumes of DNA clean up reagent (SeqType ® R24 Kit, IPE).

Purified libraries were quantified using the Qubit quantitation system and diluted to 0.008 ng/μL, from which 25 μL was taken as a readily available library for later sequencing. Emulsion PCR (using the Ion PGM template OT2 400 Kit) and subsequent positive bead enrichment and sequencing (using the Ion PGM sequencing 400 Kit) were performed according to the manufacturer's instructions.

SeqVision V1.5 software (IPE Biotechnology, Beijing, China) was used to genotype the Ion PGM FASTQ reads based on a reference sequence comparison method we developed. First, the converted FASTA reads were sorted into different sample libraries and then into different STR libraries using code sequences and upstream primers. Data filtering was then performed, by identifying prespecified STR end patterns to retain only reads that spanned the entire repeat regions. The accuracy rate decreased as read lengths were processed using the PGM platform, and more precise alignment was allowed by trimming the sequencing reads to the identified end. It is important to note that parallel trimming was performed on the preconstructed reference, as most alignment algorithms compare the coverage against the reference sequence. For qualified loci that generated adequate aligning reads, genotypes were derived based on the ratio of aligning reads between any allele to the whole locus (with alleles with >25% aligning reads called).

  Results Top

Short tandem repeat typing and NGS Sequence result

The results obtained from the D12S391 locus tested using the PowerPlex 21 system are shown in [Figure 1]. The results from all the 38 loci using both multiplex autosomal STR kits are shown in [Table 1]. The only mismatch observed was at the D12S391 locus.
Figure 1: Locus D12S391 results in the mother, child, and alleged father

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Table 1: Genotyping results for the alleged father, child, and mother at 38 short tandem repeat loci

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Allele 19.3 in the child may have been caused by a one-base loss in allele 20, inherited from one of his parents. NGS showed the loss of one adenine between the 19.3 allele of the child and allele 20 of the mother, in the 10th AGAT repeat motif, while there was no sequence variation at allele 20 between the child and the alleged father [Table 2].
Table 2: Sequence information in locus D12S391

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  Discussion Top

STRs have been widely used in human identification and kinship analysis, but a recent study has illustrated that their relatively high mutation rates are disadvantageous to forensic analysis.[3] Kinship tests are currently conducted using autosomal STR systems. When mutations are detected, they can be validated using different reagent kits containing the same locus, but ideally, sequencing should be used to directly compare the sequences. NGS can provide not only length-based genotypes, but also the full sequences from all STR loci.[4] The availability of full sequence information makes it possible to investigate the true variation within STR loci and identify previously unknown alleles and mutational events in kinship analysis.[5] In this case, D12S391 did not conform to Mendelian inheritance, prompting the use of NGS, which demonstrated that there was no difference between the child's allele 20 and the alleged father's allele 20, with the child's 19.3 due to a single-base loss inherited from the mother's allele 20. The most commonly reported microsatellite mutations are single- and multi-step mutations; however, single-base loss mutations have not been previously reported.[6],[7] The availability of full-sequence information from NGS makes it possible to investigate the true variation within STR loci and identify previously unknown alleles and mutational events in kinship analysis.


We thank Dr. YaJiao Pan for her technological assistance.

Financial support and sponsorship

This work was supported by IPE Biotechnology, who provided the SeqType ® R24 kit.

Conflicts of interest

There are no conflicts of interest.

  References Top

Dauber EM, Kratzer A, Neuhuber F, Parson W, Klintschar M, Bär W, et al. Germline mutations of STR-alleles include multi-step mutations as defined by sequencing of repeat and flanking regions. Forensic Sci Int Genet 2012;6:381-6.  Back to cited text no. 1
Xie B, Chen L, Yang Y, Lv Y, Chen J, Shi Y, et al. Genetic distribution of 39 STR loci in 1027 unrelated Han individuals from Northern China. Forensic Sci Int Genet 2015;19:205-6.  Back to cited text no. 2
Zhu WQ, Jin B, Su Q, Luo HB, Li YB, Wu J, et a l. Mutation study of 28 autosomal STR loci in Southwest Chinese Han population. Forensic Sci Int Genet Suppl Ser 2015;5:e298-9.  Back to cited text no. 3
Gettings KB, Aponte RA, Vallone PM, Butler JM. STR allele sequence variation: Current knowledge and future issues. Forensic Sci Int Genet 2015;18:118-30.  Back to cited text no. 4
Zeng X, King JL, Stoljarova M, Warshauer DH, LaRue BL, Sajantila A, et al. High sensitivity multiplex short tandem repeat loci analyses with massively parallel sequencing. Forensic Sci Int Genet 2015;16:38-47.  Back to cited text no. 5
Børsting C, Morling N. Next generation sequencing and its applications in forensic genetics. Forensic Sci Int Genet 2015;18:78-89.  Back to cited text no. 6
Dalsgaard S, Rockenbauer E, Gelardi C, Borsting C, Fordyce SL, Morling N. Characterization of mutations and sequence variations in complex STR loci by second generation sequencing. Forensic Sci Int Genet Suppl Ser 2013;4;e218-9.  Back to cited text no. 7


  [Figure 1]

  [Table 1], [Table 2]


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