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Population genetics of insertion–deletion polymorphisms in South Koreans using Investigator DIPplex kit
Forensic Science International: Genetics 8 (2014) 80–83
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Short communication
Population genetics of insertion–deletion polymorphisms in South Koreans using Investigator DIPplex kit Ki Min Seong a,b, Ji Hye Park a, Young Se Hyun b, Pil Won Kang a, Dong Ho Choi a, Myun Soo Han a, Ki Won Park a, Ki Wha Chung b,* a b
Forensic DNA Center, National Forensic Service, Seoul 158-707, Republic of Korea Department of Biological Science, Kongju National University, 182 Gongju, Chungnam 314-701, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 March 2013 Received in revised form 20 June 2013 Accepted 24 June 2013
We assessed the applicability of 30 insertion–deletion polymorphisms (INDELs) in forensic use and the level of genetic diversity in South Korea (n = 373) using the Investigator DIPplex1 kit (Qiagen). Allele frequencies, heterozygocities, and forensic efficacy parameters were determined. No deviation from Hardy–Weinberg equilibrium was observed for any of the INDEL markers. A high level of discrimination power was observed (combined power of discrimination: 0.99999999995). The combined match probability value was 2.84 1011 and the mean typical paternity indices were 0.878. Furthermore, we found one microvariant allele at HLD93 (rs2307570) that has not been reported. We expect that these 30 loci of INDEL markers will be useful for forensic identification and paternity testing in the South Korean population. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Key words: Investigator DIPplex1 kit INDEL Insertion–deletion polymorphisms Biallelic South Korean
1. Introduction The typing of short tandem repeats (STRs) is the most representative identification method in the forensic field. Although STR-based typing performs well, its limitations (relatively high amplicon size, artifacts, and mutation rates) have been well documented [1–3]. The forensic community has recently focused on alternative and supplementary genetic markers: single nucleotide polymorphisms (SNPs) and insertion–deletion polymorphisms (INDELs). SNPs can be captured in smaller amplicons than STRs, do not produce stutter during PCR, and their mutation rates are orders of magnitude lower than that of STRs [4]. To date, complex approaches, which are unwieldy and often not quantitative, have been sought [5–9]. INDELs are also widely distributed throughout the human genome [10,11], and have advantages in amplicon size [12,13], artifacts [14], and mutation rate in comparison with STRs [15]. When compared to SNPs, INDELs exhibit similar potential for forensic applications, while offering the value of a more simplified analytical process [4,16–18]. The difference between SNPs and INDELs in terms of analysis is that the latter is based on size rather than detecting a nucleotide substitution, so INDELs can be readily analyzed using capillary electrophoresis. Essentially, the analysis of INDELs is similar to that of STRs. In spite of the indisputable
advantages of INDELs, its application in routine forensic work remains rare. Several studies on INDELs have recently been published, and interest in this area has grown since the development of the first commercial kit by Qiagen (Hilden, Germany) [19–23]. The kit contains 30 INDEL markers, which are located over 19 autosomes and the amelogenin gene as a sex informative marker, with a maximum amplicon length of 160 base pairs. Here, we report polymorphisms of 30 INDEL markers using Investigator DIPplex1 kit in 373 unrelated South Korean individuals. We analyzed forensic efficiency parameters in order to evaluate forensic application of INDELs in the South Korean population. 2. Materials and methods 2.1. Samples Buccal swabs were collected from 373 unrelated individuals residing in five big cities (Seoul, Daejeon, Busan, Gwangju, and Daegu), South Korea. Written informed consent was obtained from all participants. All samples were in compliance with Korean legislation on National DNA Database. Each sample was amplified using the AmpFlSTR Identifiler1 kit (Applied Biosystems, Foster City, CA, USA) and shown to be unique. 2.2. DNA extraction, quantification and amplification
* Corresponding author. Tel.: +82 41 850 8506; fax: +82 41 850 0957. E-mail address: [email protected] (K.W. Chung). 1872-4973/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsigen.2013.06.013
Genomic DNA was isolated from buccal swabs using a QIAamp1 DNA Mini kit (Qiagen). The quantity of DNA was determined by
K.M. Seong et al. / Forensic Science International: Genetics 8 (2014) 80–83
81
Table 1 Allele frequencies and forensic parameters of 30 loci in 373 South Koreans. Markers
DIP()
DIP(+)
Ho
He
PIC
PD
MP
PE
TPI
HWE
HLD77 (rs1611048) HLD45 (rs2307959) HLD131 (rs1611001) HLD70 (rs2307652) HDL6 (rs1610905) HLD111 (rs1305047) HLD58 (rs1610937) HLD56 (rs2308292) HLD118 (rs16438) HLD92 (rs17174476) HLD93 (rs2307570) HLD99 (rs2308163) HLD88 (rs8190570) HLD101 (rs2307433) HLD67 (rs1305056) HLD83 (rs2308072) HLD114 (rs2307581) HLD48 (rs28369942) HLD124 (rs6481) HLD122 (rs8178524) HLD125 (rs16388) HLD64 (rs1610935) HLD81 (rs17879936) HLD136 (rs16363) HLD133 (rs2067235) HLD97 (rs17238892) HLD40 (rs2307956) HLD128 (rs2307924) HLD39 (rs17878444) HLD84 (rs3081400)
0.539 0.410 0.639 0.366 0.568 0.928 0.668 0.472 0.050 0.579 0.394 0.086 0.492 0.567 0.318 0.603 0.713 0.623 0.444 0.737 0.645 0.145 0.127 0.418 0.625 0.670 0.374 0.653 0.873 0.244
0.461 0.590 0.361 0.634 0.432 0.072 0.332 0.528 0.950 0.421 0.606 0.914 0.508 0.433 0.682 0.397 0.287 0.377 0.556 0.263 0.355 0.855 0.873 0.582 0.375 0.330 0.626 0.347 0.127 0.756
0.499 0.504 0.448 0.512 0.520 0.145 0.418 0.531 0.094 0.493 0.477 0.150 0.464 0.496 0.399 0.472 0.402 0.485 0.523 0.391 0.485 0.257 0.217 0.504 0.472 0.418 0.501 0.426 0.228 0.365
0.498 0.485 0.462 0.465 0.491 0.134 0.444 0.499 0.094 0.488 0.478 0.157 0.501 0.492 0.434 0.479 0.410 0.470 0.494 0.388 0.459 0.248 0.223 0.487 0.470 0.443 0.469 0.454 0.223 0.369
0.374 0.367 0.355 0.357 0.370 0.128 0.346 0.374 0.090 0.368 0.363 0.145 0.375 0.370 0.340 0.365 0.325 0.360 0.372 0.313 0.353 0.217 0.196 0.368 0.359 0.344 0.595 0.351 0.198 0.301
0.624 0.606 0.608 0.585 0.604 0.253 0.601 0.608 0.175 0.617 0.614 0.273 0.636 0.618 0.594 0.620 0.568 0.603 0.606 0.550 0.590 0.406 0.366 0.610 0.609 0.598 0.359 0.607 0.372 0.534
0.376 0.394 0.392 0.415 0.396 0.747 0.399 0.392 0.825 0.383 0.386 0.727 0.364 0.382 0.406 0.380 0.432 0.397 0.394 0.450 0.410 0.594 0.634 0.390 0.391 0.402 0.405 0.393 0.628 0.466
0.184 0.193 0.148 0.196 0.208 0.017 0.125 0.213 0.007 0.179 0.166 0.018 0.166 0.184 0.114 0.160 0.114 0.173 0.208 0.110 0.175 0.047 0.034 0.191 0.162 0.125 0.184 0.131 0.038 0.094
0.992 1.014 0.910 1.019 1.048 0.587 0.859 1.060 0.552 0.982 0.952 0.588 0.952 0.992 0.833 0.937 0.833 0.966 1.048 0.825 0.971 0.673 0.637 1.008 0.942 0.859 0.992 0.871 0.648 0.787
1.000 0.457 0.574 0.058 0.292 0.240 0.294 0.255 0.609 0.916 1.000 0.332 0.177 0.916 0.149 0.829 0.801 0.584 0.297 0.895 0.307 0.537 0.641 0.524 1.000 0.293 0.184 0.251 0.816 0.781
DIP(), deletion; DIP(+), insertion; Ho, observed heterizygocity; He, expected heterozygocity; PIC, polymorphic information contents; PD, power of discrimination; MP, matching probability; PE, power of exclusion; TPI, typical paternity
qPCR using a Quantifiler1 Quantification kit (Applied Biosystems) on an ABI 7500 Real-Time PCR System (Applied Biosystems) according to the manufacturer’s recommendations. With the Investigator DIPplex1 kit (Qiagen), 30 biallelic autosomal INDELs and amelogenin are amplified according to the manufacturer’s recommendation using a GeneAmp PCR system 9700 thermal cycler (Applied Biosystems). 2.3. INDEL analysis Amplified PCR products were subjected to capillary electrophoresis on a 3500xl Genetic Analyzer (Applied Biosystems) with 36 cm capillary arrays and POP 4 polymer, following the recommendations of the kit. Samples were analyzed using GeneMapper ID-X1 v1.2 (Applied Biosystems). 2.4. Quality control Experiments were performed in the laboratory of the Forensic DNA Center, National Forensic Service, which is accredited according to the ISO 17025 standard. 2.5. Statistical analysis Estimation of allele frequencies and heterozygosities, deviation from the Hardy–Weinberg equilibrium (HWE), the statistical significance of linkage disequilibrium (LD) among loci pairs, and the exact test of population differentiation (Fst) were calculated using Arlequin 3.5 software [24]. Forensic efficiency parameters were assessed by estimating discrimination power (DP), match probability (MP), polymorphic information content (PIC), typical paternity index (TPI), and power of exclusion (PE) using a Powerstats V12 software (Promega, Madison, WI, USA).
2.6. Sequencing analysis of microvariant Primers for sequencing at HLD93 (rs2307570) were designed using Primer3 software [25]. Forward and reverse primer sequences are 50 -AATGCCACCACTTTACTTTCAC-30 and 50 TTTTGTTGAAGAGCTTAGTCGTC-30 respectively. The sequencing reactions were performed using the BigDye Terminator1 v1.1 Cycle Sequencing kit (Applied Biosystems) according to the manufacturer’s recommendations. 3. Results and discussions INDEL-types, allele frequencies, and forensic efficiency parameters for the 30 INDEL loci in the South Korean population are shown in Table 1 and Supplementary Table 1. There was no deviation from the Hardy–Weinberg equilibrium (P > 0.05) for all 30 loci analyzed. The expected heterozgosities ranged from 0.094 (HLD118) to 0.501 (HLD88) with a mean value of 0.407, while the observed heterozygosities ranged from 0.094 (HLD118) to 0.531 (HLD56) with a mean value of 0.410. The low heterozygosity (below than 0.100) at HLD118 was similarly observed in two Asian populations [22,26]. Values for the polymorphic information content ranged between 0.090 and 0.595. Except for HLD118, all markers appeared to be highly polymorphic and sufficiently informative for forensic application with the South Korean population. Supplementary material related to this article can be found, in the online version, at doi:10.1016/j.fsigen.2013.06.013. Linkage disequilibrium for all possible combinations located on the same chromosome revealed little evidence for departures from independence after Bonferroni’s correction [27]. Therefore, these 30 INDEL markers can be treated as independent for calculation of the matching probability (Supplementary Table 2).
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K.M. Seong et al. / Forensic Science International: Genetics 8 (2014) 80–83
Fig. 1. Sequencing analysis of microvariant caused by heterozygous deletion at HLD93. (A) Electropherogram of normal (upper) and microvariant (lower) allele at HLD93. (B) Sequencing analysis of normal homozygote of insertion allele (upper) and microvariant (lower) at HLD93. ‘ACTTT’ in red box indicates main INDEL site (insertion sequence), and ‘GTTT’ in red box indicates deletion sequence in microvariant allele at HLD93.
Supplementary material related to this article can be found, in the online version, at doi:10.1016/j.fsigen.2013.06.013. In order to determine forensic efficiency, we evaluated power of discrimination (PD), power of exclusion (PE), and matching probability (MP). The combined power of discrimination (CPD) and the combined power of exclusion (CPE) for 30 INDEL markers were 0.99999999995 and 0.988, respectively. The combined matching probability was 2.84 1011 for South Koreans, allowing a satisfactory level of discrimination in forensic cases. The allelic frequencies for each locus in the South Korean population were compared with those in other ethnic groups (Supplementary Table 3). Allele frequencies of all the loci in this study were not significantly different from frequencies in other Korean study [28]. This study also revealed no significant different
allele frequencies at most loci in two Asian populations except for HLD118 (rs16438) and HLD81 (rs17879936) [22,26]. But allele frequencies of most loci in this study were significantly different from those in US-Caucasian [26], US-Hispanic [26], AfricanAmerican [26], Danish [20], Spanish [29], Basque [29], Hungarian [30], North-East Italian [19], Czech [23], German [31], Finnish [21] and Somali [21] populations (after Bonferroni’s correction: P < 0.0036). Supplementary material related to this article can be found, in the online version, at doi:10.1016/j.fsigen.2013.06.013. This study identified one microvariant allele at HLD93 (rs2307570) that has not been reported previously. This locus displayed an off-ladder allele located in a position 1-bp longer than the regular deletion allele (Fig. 1A). The sequencing analysis
K.M. Seong et al. / Forensic Science International: Genetics 8 (2014) 80–83
revealed that the off-ladder peak was a regular insertion allele with a neighboring 4-bp (GTTT) deletion located 8 bases downstream from the main INDEL site (ACTTT) (Fig. 1B). In conclusion, we expect that these 30 loci of INDEL markers will be a useful tool for forensic identification, especially in challenging DNA cases in South Korea. Although the Investigator DIPplex1 kit showed a lower combined power of exclusion (CPE) than STRs, the low mutation rate of INDEL markers have been presumed to help distinguish true exclusions in paternity testing. It will provide a complementary tool for kinship analysis with STRs, especially in cases with STR loci mutations involved. This paper follows the guidelines for publication of population data requested by the journal [32]. Acknowledgement This study was supported by a grant from the National Forensic Service (NFS), South Korea. References [1] M. Fondevila, C. Phillips, N. Navera´n, M. Cerezo, A. Rodrı´guez, R. Calvo, L. Ferna´ndez, A´. Carracedo, M. Lareu, Challenging DNA: assessment of a range genotyping approaches for highly degraded forensic samples, Forensic Sci. Int. Suppl. Ser. 1 (2008) 26–28. [2] P. Gill, J. Whitaker, C. Flaxman, N. Brown, J. Buckleton, An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA, Forensic Sci. Int. 112 (2000) 17–40. [3] A.D. Kloosterman, P. Kersbergen, Efficacy and limits of genotyping low copy number DNA samples by multiplex PCR of STR loci, Int. Congr. Ser. 1239 (2003) 795–798. [4] B. Budowle, A. van Daal, Forensically relevant SNP classes, Biotechniques 44 (2008) 603–610. [5] L.A. Dixon, C.M. Murray, E.J. Archer, A.E. Dobbins, P. Koumi, P. Gill, Validation of a 21-locus autosomal SNP multiplex for forensic identification purposes, Forensic Sci. Int. 154 (2005) 62–77. [6] K.K. Kidd, A.J. Pakstis, W.C. Speed, E.L. Grigorenko, S.L.B. Kajuna, N.J. Karoma, S. Kungulilo, J-J. Kim, R.-B. Lu, A. Odunsi, F. Okonofua, J. Parnas, L.O. Schulz, O.V. Zhukova, J.R. Kidd, Developing a SNP panel for forensic identification of individuals, Forensic Sci. Int. 164 (2006) 20–32. [7] C. Phillips, R. Fang, D. Ballard, M. Fondevila, C. Harrison, F. Hyland, E. MusgraveBrown, C. Proff, E. Ramos-Luis, B. Sobrino, A´. Carracedo, M.R. Furtado, D.S. Court, P.M. Schneider, Evaluation of the Genplex SNP typing system and a 49plex forensic marker panel, Forensic Sci. Int. Genet. 1 (2007) 180–185. [8] A-M. Divne, M. Allen, A DNA microarray system for forensic SNP analysis, Forensic Sci. Int. 154 (2005) 111–121. [9] J.J. Sanchez, C. Phillips, C. Børsting, K. Balogh, M. Bogus, M. Fondevila, C.D. Harrison, E. Musgrave-Brown, A. Salas, D. Syndercombe-Court, P.M. Schneider, A´. Carracedo, N. Morling, A multiplex assay with 52 single nucleotide polymorphisms for human identification, Electrophoresis 27 (2006) 1713–1724. [10] J.L. Weber, D. David, J. Heil, Y. Fan, C. Zhao, G. Marth, Human diallelic insertion/ deletion polymorphisms, Am. J. Hum. Genet. 71 (2002) 854–862. [11] R.E. Mills, C.T. Luttig, C.E. Larkins, A. Beauchamp, C. Tsui, W.S. Pittard, S.E. Devine, An initial map of insertion and deletion (INDEL) variation in the human genome, Genome Res. 16 (2006) 1182–1190.
83
[12] C. Hollard, F. Mendisco, C. Keyser, E. Crube´zy, B. Ludes, First application of the Investigator DIPplex indels typing kit for the analysis of ancient DNA samples, Forensic Sci. Int. Genet. Suppl. Ser. 3 (2011) 393–394. [13] C. Romanini, M.L. Catelli, A. Borosky, R. Pereira, M. Romero, M. Salado Puerto, C. Phillips, M. Fondevila, A. Freire, C. Santos, A´. Carracedo, M.V. Lareu, L. Gusma˜o, C.M. Vullo, Typing short amplicon binary polymorphisms: supplementary SNP and indel genetic information in the analysis of highly degraded skeletal remains, Forensic Sci. Int. Genet. 6 (2012) 469–476. [14] A. Carvalho, L. Caine´, R. Carvalho, M.F. Pinheiro, Application of indels (Investigator DIPplex) in mixture samples, Forensic Sci. Int. Genet. Suppl. Ser. 3 (2011) 351–352. [15] M.W. Nachman, S.L. Crowell, Estimate of the mutation rate per nucleotide in humans, Genetics 156 (2000) 297–304. [16] R. Pereira, C. Phillips, C. Alves, C. Amorim, A. Carracedo, L. Gusma˜o, A new multiplex for human identification using insertion/deletion polymorphisms, Electrophoresis 30 (2009) 3682–3690. [17] N.P. Santos, E.M. Ribeiro-Rodrigues, A.K. Ribeiro-Dos-Santos, R. Pereira, L. Gusma˜o, A. Amorim, J.F. Guerreiro, M.A. Zago, C. Matte, M.H. Hutz, S.E. Santos, Assessing individual interethnic admixture and population substructure using a 48-insertion-deletion (INSEL) ancestry-informative marker (AIM) panel, Hum. Mutat. 31 (2010) 184–190. [18] C. Li, S. Zhao, S. Zhang, L. Li, Y. Liu, J. Chen, J. Xue, Genetic polymorphism of 29 highly informative InDel markers for forensic use in the Chinese Han population, Forensic Sci. Int. Genet. 5 (2011) e27–e30. [19] S. Turrina, G. Filippini, D.D. Leo, Forensic evaluation of the Investigator DIPplex typing system, Forensic Sci. Int. Genet. Suppl. Ser. 3 (2011) e331–e332. [20] S.L. Friis, C. Børsting, E. Rockenbauer, L. Poulsen, S.F. Freslund, C. Tomas, N. Morling, Typing of 30 insertions/deletions in Danes using the first commercial indel kit-Mentipe1 DIPplex, Forensic Sci. Int. Genet. 6 (2012) e72–e74. [21] A.M. Neuvonen, J.U. Palo, M. Hedman, A. Sajantila, Discrimination power of Investigator DIPplex loci in Finnish and Somali populations, Forensic Sci. Int. Genet. 6 (2012) e99–e102. [22] B. Larue, J. Ge, J.L. King, B. Budowle, A validation study of the Qiagen Investigator DIPplex kit; an INDEL-based assay for human identification, Int. J. Legal Med. 126 (2012) 533–540. [23] A. Zidkova, A. Horinek, V. Kebrdlova, M. Korabecna, Application of the new insertion–deletion polymorphism kit for forensic identification and parentage testing on the Czech population, Int. J. Legal Med. 127 (2013) 7–10. [24] L. Excoffier, H. Lischer, Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows, Mol. Ecol. Res. 10 (2010) 564–567. [25] S. Rozen, H. Skaletsky, Primer3 on the WWW for general users and for biologist programmers, Methods Mol. Biol. 132 (2000) 365–386. [26] M. Fondevila, C. Phillips, C. Santos, R. Pereira, L. Gusma˜o, A´. Carracedo, J.M. Butler, M.V. Lareu, P.M. Vallone, Forensic performance of insertion–deletion marker assays, Int. J. Legal Med. 126 (2012) 725–737. [27] B. Weir, C.C. Cockerham, Estimating F-statistics for the analysis of population structure, Evolution 38 (1984) 1358–1370. [28] E.H. Kim, H.Y. Lee, I.S. Yang, W.I. Yang, K.-J. Shin, Population data for 30 insertion– deletion markers in a Korean population, Int. J. Legal Med. (2013), http:// dx.doi.org/10.1007/s00414-013-0851-6 (in press). [29] P. Martı´n, O. Garcı´a, B. Heinrichs, I. Yurrebaso, A. Aruirre, A. Alonso, Population genetic data of 30 autosomal indels in Central Spain and the Basque Country populations, Forensic Sci. Int. Genet. 7 (2013) e27–e30. [30] Z. Kis, A. Zala´n, A. Vo¨lgyi, Z. Kozma, L. Domja´n, H. Pamjav, Genome deletion and insertion polymorphisms (DIPs) in the Hungarian population, Forensic Sci. Int. Genet. 6 (2012) e125–e126. [31] Qiagen supplementary material: population data for Investigator DIPplex. Available at http://www.qiagen.com/literature/render.aspx?id=200253. [32] A´. Carracedo, J.M. Butler, L. Gusma˜o, A. Linacre, W. Parson, L. Roewer, P.M. Schneider, New guidelines for the population genetic population data, Forensic Sci. Int. Genet. 7 (2013) 217–220.
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Short communication
Population genetics of insertion–deletion polymorphisms in South Koreans using Investigator DIPplex kit Ki Min Seong a,b, Ji Hye Park a, Young Se Hyun b, Pil Won Kang a, Dong Ho Choi a, Myun Soo Han a, Ki Won Park a, Ki Wha Chung b,* a b
Forensic DNA Center, National Forensic Service, Seoul 158-707, Republic of Korea Department of Biological Science, Kongju National University, 182 Gongju, Chungnam 314-701, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 March 2013 Received in revised form 20 June 2013 Accepted 24 June 2013
We assessed the applicability of 30 insertion–deletion polymorphisms (INDELs) in forensic use and the level of genetic diversity in South Korea (n = 373) using the Investigator DIPplex1 kit (Qiagen). Allele frequencies, heterozygocities, and forensic efficacy parameters were determined. No deviation from Hardy–Weinberg equilibrium was observed for any of the INDEL markers. A high level of discrimination power was observed (combined power of discrimination: 0.99999999995). The combined match probability value was 2.84 1011 and the mean typical paternity indices were 0.878. Furthermore, we found one microvariant allele at HLD93 (rs2307570) that has not been reported. We expect that these 30 loci of INDEL markers will be useful for forensic identification and paternity testing in the South Korean population. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Key words: Investigator DIPplex1 kit INDEL Insertion–deletion polymorphisms Biallelic South Korean
1. Introduction The typing of short tandem repeats (STRs) is the most representative identification method in the forensic field. Although STR-based typing performs well, its limitations (relatively high amplicon size, artifacts, and mutation rates) have been well documented [1–3]. The forensic community has recently focused on alternative and supplementary genetic markers: single nucleotide polymorphisms (SNPs) and insertion–deletion polymorphisms (INDELs). SNPs can be captured in smaller amplicons than STRs, do not produce stutter during PCR, and their mutation rates are orders of magnitude lower than that of STRs [4]. To date, complex approaches, which are unwieldy and often not quantitative, have been sought [5–9]. INDELs are also widely distributed throughout the human genome [10,11], and have advantages in amplicon size [12,13], artifacts [14], and mutation rate in comparison with STRs [15]. When compared to SNPs, INDELs exhibit similar potential for forensic applications, while offering the value of a more simplified analytical process [4,16–18]. The difference between SNPs and INDELs in terms of analysis is that the latter is based on size rather than detecting a nucleotide substitution, so INDELs can be readily analyzed using capillary electrophoresis. Essentially, the analysis of INDELs is similar to that of STRs. In spite of the indisputable
advantages of INDELs, its application in routine forensic work remains rare. Several studies on INDELs have recently been published, and interest in this area has grown since the development of the first commercial kit by Qiagen (Hilden, Germany) [19–23]. The kit contains 30 INDEL markers, which are located over 19 autosomes and the amelogenin gene as a sex informative marker, with a maximum amplicon length of 160 base pairs. Here, we report polymorphisms of 30 INDEL markers using Investigator DIPplex1 kit in 373 unrelated South Korean individuals. We analyzed forensic efficiency parameters in order to evaluate forensic application of INDELs in the South Korean population. 2. Materials and methods 2.1. Samples Buccal swabs were collected from 373 unrelated individuals residing in five big cities (Seoul, Daejeon, Busan, Gwangju, and Daegu), South Korea. Written informed consent was obtained from all participants. All samples were in compliance with Korean legislation on National DNA Database. Each sample was amplified using the AmpFlSTR Identifiler1 kit (Applied Biosystems, Foster City, CA, USA) and shown to be unique. 2.2. DNA extraction, quantification and amplification
* Corresponding author. Tel.: +82 41 850 8506; fax: +82 41 850 0957. E-mail address: [email protected] (K.W. Chung). 1872-4973/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsigen.2013.06.013
Genomic DNA was isolated from buccal swabs using a QIAamp1 DNA Mini kit (Qiagen). The quantity of DNA was determined by
K.M. Seong et al. / Forensic Science International: Genetics 8 (2014) 80–83
81
Table 1 Allele frequencies and forensic parameters of 30 loci in 373 South Koreans. Markers
DIP()
DIP(+)
Ho
He
PIC
PD
MP
PE
TPI
HWE
HLD77 (rs1611048) HLD45 (rs2307959) HLD131 (rs1611001) HLD70 (rs2307652) HDL6 (rs1610905) HLD111 (rs1305047) HLD58 (rs1610937) HLD56 (rs2308292) HLD118 (rs16438) HLD92 (rs17174476) HLD93 (rs2307570) HLD99 (rs2308163) HLD88 (rs8190570) HLD101 (rs2307433) HLD67 (rs1305056) HLD83 (rs2308072) HLD114 (rs2307581) HLD48 (rs28369942) HLD124 (rs6481) HLD122 (rs8178524) HLD125 (rs16388) HLD64 (rs1610935) HLD81 (rs17879936) HLD136 (rs16363) HLD133 (rs2067235) HLD97 (rs17238892) HLD40 (rs2307956) HLD128 (rs2307924) HLD39 (rs17878444) HLD84 (rs3081400)
0.539 0.410 0.639 0.366 0.568 0.928 0.668 0.472 0.050 0.579 0.394 0.086 0.492 0.567 0.318 0.603 0.713 0.623 0.444 0.737 0.645 0.145 0.127 0.418 0.625 0.670 0.374 0.653 0.873 0.244
0.461 0.590 0.361 0.634 0.432 0.072 0.332 0.528 0.950 0.421 0.606 0.914 0.508 0.433 0.682 0.397 0.287 0.377 0.556 0.263 0.355 0.855 0.873 0.582 0.375 0.330 0.626 0.347 0.127 0.756
0.499 0.504 0.448 0.512 0.520 0.145 0.418 0.531 0.094 0.493 0.477 0.150 0.464 0.496 0.399 0.472 0.402 0.485 0.523 0.391 0.485 0.257 0.217 0.504 0.472 0.418 0.501 0.426 0.228 0.365
0.498 0.485 0.462 0.465 0.491 0.134 0.444 0.499 0.094 0.488 0.478 0.157 0.501 0.492 0.434 0.479 0.410 0.470 0.494 0.388 0.459 0.248 0.223 0.487 0.470 0.443 0.469 0.454 0.223 0.369
0.374 0.367 0.355 0.357 0.370 0.128 0.346 0.374 0.090 0.368 0.363 0.145 0.375 0.370 0.340 0.365 0.325 0.360 0.372 0.313 0.353 0.217 0.196 0.368 0.359 0.344 0.595 0.351 0.198 0.301
0.624 0.606 0.608 0.585 0.604 0.253 0.601 0.608 0.175 0.617 0.614 0.273 0.636 0.618 0.594 0.620 0.568 0.603 0.606 0.550 0.590 0.406 0.366 0.610 0.609 0.598 0.359 0.607 0.372 0.534
0.376 0.394 0.392 0.415 0.396 0.747 0.399 0.392 0.825 0.383 0.386 0.727 0.364 0.382 0.406 0.380 0.432 0.397 0.394 0.450 0.410 0.594 0.634 0.390 0.391 0.402 0.405 0.393 0.628 0.466
0.184 0.193 0.148 0.196 0.208 0.017 0.125 0.213 0.007 0.179 0.166 0.018 0.166 0.184 0.114 0.160 0.114 0.173 0.208 0.110 0.175 0.047 0.034 0.191 0.162 0.125 0.184 0.131 0.038 0.094
0.992 1.014 0.910 1.019 1.048 0.587 0.859 1.060 0.552 0.982 0.952 0.588 0.952 0.992 0.833 0.937 0.833 0.966 1.048 0.825 0.971 0.673 0.637 1.008 0.942 0.859 0.992 0.871 0.648 0.787
1.000 0.457 0.574 0.058 0.292 0.240 0.294 0.255 0.609 0.916 1.000 0.332 0.177 0.916 0.149 0.829 0.801 0.584 0.297 0.895 0.307 0.537 0.641 0.524 1.000 0.293 0.184 0.251 0.816 0.781
DIP(), deletion; DIP(+), insertion; Ho, observed heterizygocity; He, expected heterozygocity; PIC, polymorphic information contents; PD, power of discrimination; MP, matching probability; PE, power of exclusion; TPI, typical paternity
qPCR using a Quantifiler1 Quantification kit (Applied Biosystems) on an ABI 7500 Real-Time PCR System (Applied Biosystems) according to the manufacturer’s recommendations. With the Investigator DIPplex1 kit (Qiagen), 30 biallelic autosomal INDELs and amelogenin are amplified according to the manufacturer’s recommendation using a GeneAmp PCR system 9700 thermal cycler (Applied Biosystems). 2.3. INDEL analysis Amplified PCR products were subjected to capillary electrophoresis on a 3500xl Genetic Analyzer (Applied Biosystems) with 36 cm capillary arrays and POP 4 polymer, following the recommendations of the kit. Samples were analyzed using GeneMapper ID-X1 v1.2 (Applied Biosystems). 2.4. Quality control Experiments were performed in the laboratory of the Forensic DNA Center, National Forensic Service, which is accredited according to the ISO 17025 standard. 2.5. Statistical analysis Estimation of allele frequencies and heterozygosities, deviation from the Hardy–Weinberg equilibrium (HWE), the statistical significance of linkage disequilibrium (LD) among loci pairs, and the exact test of population differentiation (Fst) were calculated using Arlequin 3.5 software [24]. Forensic efficiency parameters were assessed by estimating discrimination power (DP), match probability (MP), polymorphic information content (PIC), typical paternity index (TPI), and power of exclusion (PE) using a Powerstats V12 software (Promega, Madison, WI, USA).
2.6. Sequencing analysis of microvariant Primers for sequencing at HLD93 (rs2307570) were designed using Primer3 software [25]. Forward and reverse primer sequences are 50 -AATGCCACCACTTTACTTTCAC-30 and 50 TTTTGTTGAAGAGCTTAGTCGTC-30 respectively. The sequencing reactions were performed using the BigDye Terminator1 v1.1 Cycle Sequencing kit (Applied Biosystems) according to the manufacturer’s recommendations. 3. Results and discussions INDEL-types, allele frequencies, and forensic efficiency parameters for the 30 INDEL loci in the South Korean population are shown in Table 1 and Supplementary Table 1. There was no deviation from the Hardy–Weinberg equilibrium (P > 0.05) for all 30 loci analyzed. The expected heterozgosities ranged from 0.094 (HLD118) to 0.501 (HLD88) with a mean value of 0.407, while the observed heterozygosities ranged from 0.094 (HLD118) to 0.531 (HLD56) with a mean value of 0.410. The low heterozygosity (below than 0.100) at HLD118 was similarly observed in two Asian populations [22,26]. Values for the polymorphic information content ranged between 0.090 and 0.595. Except for HLD118, all markers appeared to be highly polymorphic and sufficiently informative for forensic application with the South Korean population. Supplementary material related to this article can be found, in the online version, at doi:10.1016/j.fsigen.2013.06.013. Linkage disequilibrium for all possible combinations located on the same chromosome revealed little evidence for departures from independence after Bonferroni’s correction [27]. Therefore, these 30 INDEL markers can be treated as independent for calculation of the matching probability (Supplementary Table 2).
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Fig. 1. Sequencing analysis of microvariant caused by heterozygous deletion at HLD93. (A) Electropherogram of normal (upper) and microvariant (lower) allele at HLD93. (B) Sequencing analysis of normal homozygote of insertion allele (upper) and microvariant (lower) at HLD93. ‘ACTTT’ in red box indicates main INDEL site (insertion sequence), and ‘GTTT’ in red box indicates deletion sequence in microvariant allele at HLD93.
Supplementary material related to this article can be found, in the online version, at doi:10.1016/j.fsigen.2013.06.013. In order to determine forensic efficiency, we evaluated power of discrimination (PD), power of exclusion (PE), and matching probability (MP). The combined power of discrimination (CPD) and the combined power of exclusion (CPE) for 30 INDEL markers were 0.99999999995 and 0.988, respectively. The combined matching probability was 2.84 1011 for South Koreans, allowing a satisfactory level of discrimination in forensic cases. The allelic frequencies for each locus in the South Korean population were compared with those in other ethnic groups (Supplementary Table 3). Allele frequencies of all the loci in this study were not significantly different from frequencies in other Korean study [28]. This study also revealed no significant different
allele frequencies at most loci in two Asian populations except for HLD118 (rs16438) and HLD81 (rs17879936) [22,26]. But allele frequencies of most loci in this study were significantly different from those in US-Caucasian [26], US-Hispanic [26], AfricanAmerican [26], Danish [20], Spanish [29], Basque [29], Hungarian [30], North-East Italian [19], Czech [23], German [31], Finnish [21] and Somali [21] populations (after Bonferroni’s correction: P < 0.0036). Supplementary material related to this article can be found, in the online version, at doi:10.1016/j.fsigen.2013.06.013. This study identified one microvariant allele at HLD93 (rs2307570) that has not been reported previously. This locus displayed an off-ladder allele located in a position 1-bp longer than the regular deletion allele (Fig. 1A). The sequencing analysis
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revealed that the off-ladder peak was a regular insertion allele with a neighboring 4-bp (GTTT) deletion located 8 bases downstream from the main INDEL site (ACTTT) (Fig. 1B). In conclusion, we expect that these 30 loci of INDEL markers will be a useful tool for forensic identification, especially in challenging DNA cases in South Korea. Although the Investigator DIPplex1 kit showed a lower combined power of exclusion (CPE) than STRs, the low mutation rate of INDEL markers have been presumed to help distinguish true exclusions in paternity testing. It will provide a complementary tool for kinship analysis with STRs, especially in cases with STR loci mutations involved. This paper follows the guidelines for publication of population data requested by the journal [32]. Acknowledgement This study was supported by a grant from the National Forensic Service (NFS), South Korea. References [1] M. Fondevila, C. Phillips, N. Navera´n, M. Cerezo, A. Rodrı´guez, R. Calvo, L. Ferna´ndez, A´. Carracedo, M. Lareu, Challenging DNA: assessment of a range genotyping approaches for highly degraded forensic samples, Forensic Sci. Int. Suppl. Ser. 1 (2008) 26–28. [2] P. Gill, J. Whitaker, C. Flaxman, N. Brown, J. Buckleton, An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA, Forensic Sci. Int. 112 (2000) 17–40. [3] A.D. Kloosterman, P. Kersbergen, Efficacy and limits of genotyping low copy number DNA samples by multiplex PCR of STR loci, Int. Congr. Ser. 1239 (2003) 795–798. [4] B. Budowle, A. van Daal, Forensically relevant SNP classes, Biotechniques 44 (2008) 603–610. [5] L.A. Dixon, C.M. Murray, E.J. Archer, A.E. Dobbins, P. Koumi, P. Gill, Validation of a 21-locus autosomal SNP multiplex for forensic identification purposes, Forensic Sci. Int. 154 (2005) 62–77. [6] K.K. Kidd, A.J. Pakstis, W.C. Speed, E.L. Grigorenko, S.L.B. Kajuna, N.J. Karoma, S. Kungulilo, J-J. Kim, R.-B. Lu, A. Odunsi, F. Okonofua, J. Parnas, L.O. Schulz, O.V. Zhukova, J.R. Kidd, Developing a SNP panel for forensic identification of individuals, Forensic Sci. Int. 164 (2006) 20–32. [7] C. Phillips, R. Fang, D. Ballard, M. Fondevila, C. Harrison, F. Hyland, E. MusgraveBrown, C. Proff, E. Ramos-Luis, B. Sobrino, A´. Carracedo, M.R. Furtado, D.S. Court, P.M. Schneider, Evaluation of the Genplex SNP typing system and a 49plex forensic marker panel, Forensic Sci. Int. Genet. 1 (2007) 180–185. [8] A-M. Divne, M. Allen, A DNA microarray system for forensic SNP analysis, Forensic Sci. Int. 154 (2005) 111–121. [9] J.J. Sanchez, C. Phillips, C. Børsting, K. Balogh, M. Bogus, M. Fondevila, C.D. Harrison, E. Musgrave-Brown, A. Salas, D. Syndercombe-Court, P.M. Schneider, A´. Carracedo, N. Morling, A multiplex assay with 52 single nucleotide polymorphisms for human identification, Electrophoresis 27 (2006) 1713–1724. [10] J.L. Weber, D. David, J. Heil, Y. Fan, C. Zhao, G. Marth, Human diallelic insertion/ deletion polymorphisms, Am. J. Hum. Genet. 71 (2002) 854–862. [11] R.E. Mills, C.T. Luttig, C.E. Larkins, A. Beauchamp, C. Tsui, W.S. Pittard, S.E. Devine, An initial map of insertion and deletion (INDEL) variation in the human genome, Genome Res. 16 (2006) 1182–1190.
83
[12] C. Hollard, F. Mendisco, C. Keyser, E. Crube´zy, B. Ludes, First application of the Investigator DIPplex indels typing kit for the analysis of ancient DNA samples, Forensic Sci. Int. Genet. Suppl. Ser. 3 (2011) 393–394. [13] C. Romanini, M.L. Catelli, A. Borosky, R. Pereira, M. Romero, M. Salado Puerto, C. Phillips, M. Fondevila, A. Freire, C. Santos, A´. Carracedo, M.V. Lareu, L. Gusma˜o, C.M. Vullo, Typing short amplicon binary polymorphisms: supplementary SNP and indel genetic information in the analysis of highly degraded skeletal remains, Forensic Sci. Int. Genet. 6 (2012) 469–476. [14] A. Carvalho, L. Caine´, R. Carvalho, M.F. Pinheiro, Application of indels (Investigator DIPplex) in mixture samples, Forensic Sci. Int. Genet. Suppl. Ser. 3 (2011) 351–352. [15] M.W. Nachman, S.L. Crowell, Estimate of the mutation rate per nucleotide in humans, Genetics 156 (2000) 297–304. [16] R. Pereira, C. Phillips, C. Alves, C. Amorim, A. Carracedo, L. Gusma˜o, A new multiplex for human identification using insertion/deletion polymorphisms, Electrophoresis 30 (2009) 3682–3690. [17] N.P. Santos, E.M. Ribeiro-Rodrigues, A.K. Ribeiro-Dos-Santos, R. Pereira, L. Gusma˜o, A. Amorim, J.F. Guerreiro, M.A. Zago, C. Matte, M.H. Hutz, S.E. Santos, Assessing individual interethnic admixture and population substructure using a 48-insertion-deletion (INSEL) ancestry-informative marker (AIM) panel, Hum. Mutat. 31 (2010) 184–190. [18] C. Li, S. Zhao, S. Zhang, L. Li, Y. Liu, J. Chen, J. Xue, Genetic polymorphism of 29 highly informative InDel markers for forensic use in the Chinese Han population, Forensic Sci. Int. Genet. 5 (2011) e27–e30. [19] S. Turrina, G. Filippini, D.D. Leo, Forensic evaluation of the Investigator DIPplex typing system, Forensic Sci. Int. Genet. Suppl. Ser. 3 (2011) e331–e332. [20] S.L. Friis, C. Børsting, E. Rockenbauer, L. Poulsen, S.F. Freslund, C. Tomas, N. Morling, Typing of 30 insertions/deletions in Danes using the first commercial indel kit-Mentipe1 DIPplex, Forensic Sci. Int. Genet. 6 (2012) e72–e74. [21] A.M. Neuvonen, J.U. Palo, M. Hedman, A. Sajantila, Discrimination power of Investigator DIPplex loci in Finnish and Somali populations, Forensic Sci. Int. Genet. 6 (2012) e99–e102. [22] B. Larue, J. Ge, J.L. King, B. Budowle, A validation study of the Qiagen Investigator DIPplex kit; an INDEL-based assay for human identification, Int. J. Legal Med. 126 (2012) 533–540. [23] A. Zidkova, A. Horinek, V. Kebrdlova, M. Korabecna, Application of the new insertion–deletion polymorphism kit for forensic identification and parentage testing on the Czech population, Int. J. Legal Med. 127 (2013) 7–10. [24] L. Excoffier, H. Lischer, Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows, Mol. Ecol. Res. 10 (2010) 564–567. [25] S. Rozen, H. Skaletsky, Primer3 on the WWW for general users and for biologist programmers, Methods Mol. Biol. 132 (2000) 365–386. [26] M. Fondevila, C. Phillips, C. Santos, R. Pereira, L. Gusma˜o, A´. Carracedo, J.M. Butler, M.V. Lareu, P.M. Vallone, Forensic performance of insertion–deletion marker assays, Int. J. Legal Med. 126 (2012) 725–737. [27] B. Weir, C.C. Cockerham, Estimating F-statistics for the analysis of population structure, Evolution 38 (1984) 1358–1370. [28] E.H. Kim, H.Y. Lee, I.S. Yang, W.I. Yang, K.-J. Shin, Population data for 30 insertion– deletion markers in a Korean population, Int. J. Legal Med. (2013), http:// dx.doi.org/10.1007/s00414-013-0851-6 (in press). [29] P. Martı´n, O. Garcı´a, B. Heinrichs, I. Yurrebaso, A. Aruirre, A. Alonso, Population genetic data of 30 autosomal indels in Central Spain and the Basque Country populations, Forensic Sci. Int. Genet. 7 (2013) e27–e30. [30] Z. Kis, A. Zala´n, A. Vo¨lgyi, Z. Kozma, L. Domja´n, H. Pamjav, Genome deletion and insertion polymorphisms (DIPs) in the Hungarian population, Forensic Sci. Int. Genet. 6 (2012) e125–e126. [31] Qiagen supplementary material: population data for Investigator DIPplex. Available at http://www.qiagen.com/literature/render.aspx?id=200253. [32] A´. Carracedo, J.M. Butler, L. Gusma˜o, A. Linacre, W. Parson, L. Roewer, P.M. Schneider, New guidelines for the population genetic population data, Forensic Sci. Int. Genet. 7 (2013) 217–220.