Research paper
A newly devised multiplex assay of novel polymorphic non-CODIS STRs as a valuable tool for forensic application

https://doi.org/10.1016/j.fsigen.2020.102341Get rights and content

Highlights

  • We firstly discovered 18 new autosomal non-CODIS STR loci (D1S1616, D1S1608, D2S437, D3S2457, D4S2406, D4S3249, D5S2843, D5S2501, D6S1010, D8S1039, D12S1301, D14S586, D15S815, SHGC-145653, CHLC.GATA14D12, D1S1603, HUMUT7148, and CHLC.GATA84D07) by web scanning and experimental screening.

  • We developed a novel multiplex typing system named “SiFaSTR 21plex_NCII Typing System” comprising of 1) the 18 non-CODISS autosomal STRs mentioned above, 2) a CODIS locus of D2S1338, and 3) Amelogenin and DYS391.

  • The developmental validation demonstrated that SiFaSTR 21plex_NCII Typing System is an accurate, specific, sensitive and powerful tool for human DNA profiling.

  • SiFaSTR 21plex_NCII Typing System has potential to enhance the level of confidence in conclusions in complex kinship tests and benefit the forensic community.

Abstract

DNA profiling that relies on sets of highly polymorphic autosomal STR markers is widely used in the forensic field for human identification and paternity testing. However, the number of markers that are included in the STR kits that are currently available is insufficient to conclusively prove or disprove a relationship between individuals, especially when complex family scenarios are suspected or indirect analyses are required. In these cases, it becomes necessary to increase the number of loci under analysis to reach an adequate likelihood ratio (LR). In this study, we discovered 18 new autosomal non-CODIS STR loci (D1S1616, D1S1608, D2S437, D3S2457, D4S2406, D4S3249, D5S2843, D5S2501, D6S1010, D8S1039, D12S1301, D14S586, D15S815, SHGC-145653, CHLC.GATA14D12, D1S1603, HUMUT7148, and CHLC.GATA84D07) by web scanning and experimental screening. On the basis of this discovery, we developed a novel multiplex typing system named the “SiFaSTR 21plex_NCII Typing System” comprising 1) the 18 non-CODIS autosomal STRs mentioned above, 2) a CODIS locus of D2S1338, and 3) Amelogenin and DYS391. A forensic developmental validation, including sensitivity, species specificity, concordance, reproducibility, sample suitability, testing stability, and mixture testing, was performed following SWGDAM. The results of the validation studies indicated that this system is accurate, reliable and suitable for human DNA profiling. The sensitivity study of the system demonstrated that a full profile was obtainable with DNA as low as 125 pg. Species specificity was proven by the lack of cross-reactivity with a series of common animal species. The stability study demonstrated that 1 ng of control DNA could be fully genotyped with concentrations of haematin ≤ 150 μM, indigotin ≤ 5000 ng/μl, urea ≤ 16000 ng/μl, nigrosine ≤ 100 ng/μl and humic acid ≤ 20 ng/μl. In the mixture test, all of the minor alleles could be called at mixed ratios of 1:1, 1:3 and 3:1. We also investigated the allelic frequencies and forensic parameters of the included markers in 259 Chinese Han individuals. The forensic efficiency parameters, including the total power of discrimination (TDP) and the combined exclusion power in duos (CPEduos) and in trios (CPEtrios) of the system were calculated to be greater than 0.9999999, 0.9997347 and 0.9999997, respectively. This result proved that the system is suitable for human identification and paternity testing. The 18 newly discovered non-CODIS STRs and the developed system will be a valuable supplementary tool for the forensic community and will help solve complex paternity cases, evolutionary studies and population investigations.

Introduction

The study of genetic markers allows a better understanding of the history and diversity of humans and provides a technique for forensic application [[1], [2], [3], [4]]. Short tandem repeats (STRs) are common genetic markers used in forensic DNA typing and were first used in 1991 in a forensic case [5]. Capillary electrophoresis (CE) technology allows more than two dozen STRs to be profiled at once, providing DNA evidence for personal identification, paternity and relationship-testing applications [6,7]. A variety of commercially available autosomal STR kits were developed in the forensic community, which mainly detect STR loci from the databases of the Combined DNA Index System (CODIS), Interpol Standard of Loci (ISSL) and European Standard Set (ESS) [[8], [9], [10]]. Supplementary Table S1 lists the main commercialized STR kits. Twenty-four autosomal STR loci from the above databases are mainly included in these kits, which are widely used for forensic applications.

Decades ago, many Chinese families were forced to send their children away, and their families became fragmented due to difficult periods such as “the great famine” or “the great cultural revolution”. Currently, these parents want to find their “lost” children, and brothers and sisters want to find their “lost” siblings. Therefore, the demand for resolving complex parentage testing in China’s forensic community is rising. However, available commercial STR kits cannot provide enough STR markers for such cases and often result in low likelihood ratio (LR) calculations [[11], [12], [13]]. In these cases, it is becoming necessary to increase the number of loci analysed to reach an adequate number of LRs [[11], [12], [13]].

In the present study, non-CODIS STR kits such as AGCU 21 + 1 (AGCU ScienTech Incorporation, China) [14], Goldeneye DNA ID 22NC (Goldeneye Technology Ltd., China) [15,16] and Microreader 23sp ID (Suzhou Microread Genetics, China) [17] are available. Some of the non-CODIS STRs included in the kits above are less polymorphic in the Chinese population and are physically close to the above twenty-four autosomal STRs that are commonly used [9,10,[14], [15], [16], [17]]. Kits including non-CODIS STRs that are based on massively parallel sequencing (MPS) technology, such as Precision ID GlobalFiler NGS STR (20 CODIS STRs and nine non-CODIS STRs) (Thermo Fisher Scientific, Waltham, USA) [18] and ForenSeq DNA Signature Prep kit (20 CODIS STRs, seven non-CODIS STRs, 24 Y-STRs, seven X-STRs and 94 identity informative SNPs) (Verogen, San Diego, USA) [19], can also be utilized to increase the power of kinship testing. It is expensive to establish these systems, which partially explains their uncommon application in laboratories. In light of the above, we try to discover more polymorphic, independent, suitable non-CODIS STRs and, on the basis of these STRs, develop a novel multiplex typing system to provide a new tool for DNA analysis in the forensic community. The developmental validation references the following: “Validation Guidelines for DNA Analysis Methods” [20] and “Basic Quality Requirements of Forensic Science Human Fluorescent STR Multiplex PCR Testing Reagent (GA/T815-2009)” [21].

Section snippets

STR candidate selection

The screening of candidates consists of two steps: (1) web scanning and (2) experimental screening.

Construction and optimization of the novel non-CODIS multiplex system

Web scanning and experimental screening produced 18 promising polymorphic non-CODIS autosomal STRs (D1S1616, D1S1608, D2S437, D3S2457, D4S2406, D4S3249, D5S2843, D5S2501, D6S1010, D8S1039, D12S1301, D14S586, D15S815, SHGC-145653, CHLC.GATA14D12, D1S1603, HUMUT7148, and CHLC.GATA84D07). Since there are no studies or documents that describe the repeat motifs of these markers, we followed the rules in [37,38] to name the alleles of the 18 STRs. The detailed genetic information and motif repeats

Conclusion

This article outlines the development and validation of a novel multiplex system named the SiFaSTR 21plex_NCII Typing System. First, through web scanning and experimental screening, we discovered 18 new autosomal non-CODIS STR loci (D1S1616, D1S1608, D2S437, D3S2457, D4S2406, D4S3249, D5S2843, D5S2501, D6S1010, D8S1039, D12S1301, D14S586, D15S815, SHGC-145653, CHLC.GATA14D12, D1S1603, HUMUT7148, and CHLC.GATA84D07). The 18 newly discovered autosomal STRs are informative in the Chinese Han

Declaration of Competing Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This study was supported by grants from the National Natural Science Fund of China (Nos. 81625013 and 81930056), and the Shanghai Science and Technology Innovation Fund (18DZ1200300 and 19DZ2201400).

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