Utility of ForenSeq™ DNA Signature Prep Kit in the research of pairwise 2nd-degree kinship identification
The scope of forensic kinship analysis is being extended to more distant or complex relationships. However, current methods and standards in this field do not meet the needs of casework. The next-generation sequencing (NGS) technology may hold an advantage in this field to traditional methods due to its strong power to get much more genetic information. To evaluate the effectiveness of NGS to identify the 2nd-degree kinship pairs, DNA samples of 227 individuals from 49 Hebei Han pedigrees were tested by Goldeneye™ 20A kit using capillary electrophoresis (CE) to confirm the relationships within each pedigree, and those of 111 individuals within 97 confirmed grandparent-grandchild or avuncular pairs were analyzed by ForenSeq™ DNA Signature Prep Kit using MiSeq® FGx™ DNA sequencing platform. We calculated the likelihood ratio (LR) based on ITO method and the identical by state (IBS) score of 97 kinship pairs and compared with those of 97 unrelated pairs. According to the results summarized and analyzed by Fisher discriminant analysis and leave-one-out cross-validation (LOOCV) method, ITO method showed higher accuracy than IBS method, even with less information. Therefore, we proposed a recommendation of the thresholds for pairwise 2nd-degree kinship identification for Hebei Han population based on ITO method. When using ITO method based on 94 SNPs and the length information of 27 autosomal STRs, cumulative likelihood ratio (CLR) > 1 and CLR < 0.1 are recommended as the thresholds of confirming and excluding, respectively. The accuracy applying such thresholds is greater than 95%, indicating the promising application value of NGS in this field and providing a direction for further kinship identification strategy selection. Further studies are needed to get the population genetic data of loci contained in the kit based on all sequence information including flanking regions to make full use of the NGS data to improve the accuracy of kinship analysis.
Keywords2nd-degree kinship identification MiSeq® FGx™ sequencing platform ForenSeq™ DNA signature prep kit ITO method IBS method
SL and BC designed the study. MX, ZC, and QL recruited family samples and extracted DNA. MX and QD carried out CE test and NGS test. GM and LF performed data analysis. GM and SL wrote the first and last drafts of the manuscript, and all authors made critical revisions. SL and BC take responsibility for the integrity of the data and accuracy of the data analysis.
This work was supported by the National Natural Science Foundation of China (81671875); and Natural Science Foundation of Hebei Province (H2017206351).
Compliance with ethical standards
The ethical consent was obtained from the ethics committee of Hebei Medical University and statements on informed consent were signed by the adult volunteers and the parents of minors before the collection of blood samples
Disclosure of potential conflict of interest
The authors declare that they have no conflicts of interest.
- 7.Ranajit CN, Stivers DN, Birg S et al (1999) The utility of short tandem repeat loci beyond human identification: implications for development of new DNA typing systems. Electrophoresis 20:1682–1696. https://doi.org/10.1002/(SICI)1522-2683(19990101)20:8<1682::AID-ELPS1682>3.0.CO;2-Z CrossRefGoogle Scholar
- 10.Vergen (2018) ForenSeq™ DNA Signature Prep Reference Guide. https://verogen.com/wpcontent/uploads/2018/08/ForenSeq-DNA-Prep-Guide-VD2018005-A.pdf
- 13.Wang J, Huang Y, Zhang Q et al (2012) The developmental validation of the homemade GoldeneyeTM 20A PCR amplification kit. Chin J Forensic Med 27:12–15Google Scholar
- 15.Gjertson DW, Brenner CH, Baur MP, Carracedo A, Guidet F, Luque JA, Lessig R, Mayr WR, Pascali VL, Prinz M, Schneider PM, Morling N (2007) ISFG: recommendations on biostatistics in paternity testing. Forensic Sci Int Genet 1:223–231. https://doi.org/10.1016/j.fsigen.2007.06.006 CrossRefGoogle Scholar
- 16.Lu HL, Zhou KW, Lü DJ et al (2009) Application of ITO method and discriminant functions in full sibling and half sibling identification. J Forensic Med 25:118–122. https://doi.org/10.3969/j.issn.1004-5619.2009.02.012 Google Scholar
- 18.Huiling L, Qingen Y (2002) ITO method to calculate the chance of blood relationship between two individuals. Chinese J Forensic Med 17:188–191Google Scholar
- 27.Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, Smith HO, Yandell M, Evans CA, Holt RA, Gocayne JD, Amanatides P, Ballew RM, Huson DH, Wortman JR, Zhang Q, Kodira CD, Zheng XH, Chen L, Skupski M, Subramanian G, Thomas PD, Zhang J, Gabor Miklos GL, Nelson C, Broder S, Clark AG, Nadeau J, McKusick VA, Zinder N, Levine AJ, Roberts RJ, Simon M, Slayman C, Hunkapiller M, Bolanos R, Delcher A, Dew I, Fasulo D, Flanigan M, Florea L, Halpern A, Hannenhalli S, Kravitz S, Levy S, Mobarry C, Reinert K, Remington K, Abu-Threideh J, Beasley E, Biddick K, Bonazzi V, Brandon R, Cargill M, Chandramouliswaran I, Charlab R, Chaturvedi K, Deng Z, Francesco VD, Dunn P, Eilbeck K, Evangelista C, Gabrielian AE, Gan W, Ge W, Gong F, Gu Z, Guan P, Heiman TJ, Higgins ME, Ji RR, Ke Z, Ketchum KA, Lai Z, Lei Y, Li Z, Li J, Liang Y, Lin X, Lu F, Merkulov GV, Milshina N, Moore HM, Naik AK, Narayan VA, Neelam B, Nusskern D, Rusch DB, Salzberg S, Shao W, Shue B, Sun J, Wang ZY, Wang A, Wang X, Wang J, Wei MH, Wides R, Xiao C, Yan C, Yao A, Ye J, Zhan M, Zhang W, Zhang H, Zhao Q, Zheng L, Zhong F, Zhong W, Zhu SC, Zhao S, Gilbert D, Baumhueter S, Spier G, Carter C, Cravchik A, Woodage T, Ali F, An H, Awe A, Baldwin D, Baden H, Barnstead M, Barrow I, Beeson K, Busam D, Carver A, Center A, Cheng ML, Curry L, Danaher S, Davenport L, Desilets R, Dietz S, Dodson K, Doup L, Ferriera S, Garg N, Gluecksmann A, Hart B, Haynes J, Haynes C, Heiner C, Hladun S, Hostin D, Houck J, Howland T, Ibegwam C, Johnson J, Kalush F, Kline L, Koduru S, Love A, Mann F, May D, McCawley S, McIntosh T, McMullen I, Moy M, Moy L, Murphy B, Nelson K, Pfannkoch C, Pratts E, Puri V, Qureshi H, Reardon M, Rodriguez R, Rogers YH, Romblad D, Ruhfel B, Scott R, Sitter C, Smallwood M, Stewart E, Strong R, Suh E, Thomas R, Tint NN, Tse S, Vech C, Wang G, Wetter J, Williams S, Williams M, Windsor S, Winn-Deen E, Wolfe K, Zaveri J, Zaveri K, Abril JF, Guigó R, Campbell MJ, Sjolander KV, Karlak B, Kejariwal A, Mi H, Lazareva B, Hatton T, Narechania A, Diemer K, Muruganujan A, Guo N, Sato S, Bafna V, Istrail S, Lippert R, Schwartz R, Walenz B, Yooseph S, Allen D, Basu A, Baxendale J, Blick L, Caminha M, Carnes-Stine J, Caulk P, Chiang YH, Coyne M, Dahlke C, Mays AD, Dombroski M, Donnelly M, Ely D, Esparham S, Fosler C, Gire H, Glanowski S, Glasser K, Glodek A, Gorokhov M, Graham K, Gropman B, Harris M, Heil J, Henderson S, Hoover J, Jennings D, Jordan C, Jordan J, Kasha J, Kagan L, Kraft C, Levitsky A, Lewis M, Liu X, Lopez J, Ma D, Majoros W, McDaniel J, Murphy S, Newman M, Nguyen T, Nguyen N, Nodell M, Pan S, Peck J, Peterson M, Rowe W, Sanders R, Scott J, Simpson M, Smith T, Sprague A, Stockwell T, Turner R, Venter E, Wang M, Wen M, Wu D, Wu M, Xia A, Zandieh A, Zhu X (2001) The sequence of the human genome. Science 291:1304–1351. https://doi.org/10.1126/science.1058040 CrossRefGoogle Scholar
- 30.Odriozola A, Aznar JM, Valverde L, Cardoso S, Bravo ML, Builes JJ, Martínez B, Sanchez D, González-Andrade F, Sarasola E, González-Fernández MC, Jarreta BM, de Pancorbo MM (2009) SNPSTR rs59186128-D7S820 polymorphism distribution in European Caucasoid, Hispanic, and Afro-American populations. Int J Legal Med 123:527–533. https://doi.org/10.1007/s00414-009-0370-7 CrossRefGoogle Scholar
- 31.Morimoto C, Manabe S, Kawaguchi T, Kawai C, Fujimoto S, Hamano Y, Yamada R, Matsuda F, Tamaki K (2016) Pairwise kinship analysis by the index of chromosome sharing using high-density single nucleotide polymorphisms. PLoS One 11:e0160287. https://doi.org/10.1371/journal.pone.0160287 CrossRefGoogle Scholar