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The validation study of a novel assay with 30 slow and moderate mutation Y-STR markers for criminal investigation and database applications

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Abstract

The Y chromosome short tandem repeat (Y-STR) haplotyping method has been widely used in forensic applications. However, the existing Y-STR panels are not the ideal tools for criminal investigation and database applications because of their relatively low discriminatory capacity (DC) or high mutation rates. In the present study, the multiplex PCR assay (AGCU Y30) for simultaneous amplification of 30 slowly and moderately mutated Y-STR loci labeled by 6-dye fluorescence was developed and validated. The AGCU Y30 assay was capable of amplification purified DNA from casework and database samples on FTA™ cards in direct amplification module with a 10 μL reaction volume. Furthermore, the genetic diversities and forensic parameters of AGCU Y30 were performed using 719 unrelated male samples, demonstrating its high level of genetic polymorphisms and DC in Nantong Han population. This validation study demonstrated good sensitivity, mixture samples, inhibitor tolerance, precision, and concordance for the AGCU Y30, which is suitable for forensic investigation and database construction.

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Funding

This project was supported by the National Natural Science Foundation of China (NSFC, No. 81525015, 81772031), GDUPS (2017).

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Authors and Affiliations

  1. Department of Forensic Genetics, School of Forensic Medicine, Southern Medical University, Guangzhou, People’s Republic of China

    Yongsong Zhou, Tong Xie, Qiong Lan, Yating Fang & Bofeng Zhu

  2. Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, People’s Republic of China

    Yuxin Guo, Xiaoye Jin & Bofeng Zhu

  3. Clinical Research Center of Shaanxi Province for Dental and Maxillofacial Diseases, College of Stomatology, Xi’an Jiaotong University, Xi’an, People’s Republic of China

    Yuxin Guo, Xiaoye Jin & Bofeng Zhu

  4. College of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, People’s Republic of China

    Xinglin Mei

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  1. Yongsong Zhou
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Correspondence to Bofeng Zhu.

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Blood samples were collected with written informed consent and approved by the Ethics Committee of Southern Medical University.

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Electronic supplementary material

Supplementary Fig. 1

Representative electropherograms of the AGCU Y30 assay species specificity study obtained from the human, non-human and microorganism DNA samples. The templates were human (1 ng), non-human animals (2 ng each), and pooled microorganisms (10 ng). A small amount of cross-reactivity was occurred only in the chimpanzee sample. All samples were amplified on GeneAmp® PCR System 9700 thermal cycler and detected on 3500xl Genetic Analyzer. (JPG 3678 kb)

Supplementary Fig. 2

Profile of a Male:Male DNA mixture sample at the ratio of 1:9 (total of 1 ng input DNA). Mixture sample was amplified on GeneAmp® 9700, and 1 μL of each amplified product was electrophoresed on a 3500xl Genetic Analyzer using a 2 kV, 8 s injection. The minor alleles of DYS527a,b and DYS522 were dropped out and indicated by the red arrows. (JPG 469 kb)

Supplementary Fig. 3

Profile of a Male:Female DNA mixture sample containing 62.5 pg of male DNA and 500 ng of female DNA. Mixture sample was amplified on GeneAmp® 9700, and 1 μL of each amplified product was electrophoresed on a 3500xl Genetic Analyzer using a 2 kV, 8 s injection. (JPG 437 kb)

Supplementary Fig. 4

Size precision testing. AGCU Y30 assay Allelic Ladder was injected in 24 capillaries on 3500xl Genetic Analyzer. 1 μL of Allelic Ladder was electrophoresed on a 3500xl Genetic Analyzer using a 2 kV, 8 s injection. (JPG 1712 kb)

Supplementary Fig. 5

Representative Control DNA 9948 profile amplified with AGCU Y30 assay for 28, 29, 30, 31 and 32 cycles. 1 μL of PCR product was electrophoresed on a 3500xl Genetic Analyzer using a 2 kV, 8 s injection. Each cycle number profile was displayed with combine dyes in one line. (JPG 406 kb)

Supplementary Fig. 6

Representative Control DNA 9948 profile amplified with AGCU Y30 assay for 32 cycles. 1 μL of PCR product was electrophoresed on a 3500xl Genetic Analyzer using a 2 kV, 8 s injection. (JPG 420 kb)

Supplementary Fig. 7

Annealing temperature study. 1.0 ng of Control DNA 9948 amplified with AGCU Y30 assay for 30 cycles. Full profiles were obtained at the annealing temperature of 54, 56, 58, 60, and 62 °C. DYS385a,b was observed intra-locus imbalance at the annealing temperature of 62 °C. (JPG 406 kb)

Supplementary Fig. 8

Intro-color peak height ratio analysis. 1 ng of male Control DNA 9948 amplified with AGCU Y30 assay at temperatures of 54, 56, 58, 60 and 62 °C. Significant variation of intra-color balance was not observed at different annealing temperatures. (JPG 86 kb)

Supplementary Fig. 9

Reaction volumes analysis of the AGCU Y30 assay. Control DNA 9948 profile amplified with 5, 10, 15, 20, or 25 μL total reaction volume. 1 μL of PCR product was electrophoresed on a 3500xl Genetic Analyzer using a 2 kV, 8 s injection. Each reaction volume profile was displayed with combine dyes in one line. (JPG 399 kb)

Supplementary Fig. 10

Reaction components analysis. 1 ng of male Control DNA 9948 was amplified by various final concentration (0.75 ×, 1 ×, 1.25 ×) of primer set, master mix and Taq polymerase, respectively. 1 μL of PCR product was electrophoresed on a 3500xl Genetic Analyzer using a 2 kV, 8 s injection. Complete profiles were observed for all samples. (JPG 3845 kb)

Supplementary Table 1

General information of 30 Y-STRs and their genotyping results of Control DNA (DOCX 18 kb)

Supplementary Table 2

The haplotype frequency for AGCU Y30 assay. Each haplotype frequency was calculated form the genotyping results of 719 Nantong Han unrelated male samples. (XLSX 111 kb)

Supplementary Table 3

The allelic frequencies and gene diversity values for AGCU Y30 assay. Each allele frequencies and gene diversity values were calculated form the genotyping results of 719 Nantong Han unrelated male samples. (XLSX 17 kb)

Supplementary Table 4

Consistency comparison analysis with Yfiler. A set of 719 Nantong Han unrelated male samples were amplified by AGCU Y30 assay and Yfiler Kit, respectively. 1 μL of each amplified product was electrophoresed on a 3500xl Genetic Analyzer using a 2 kV, 8 s injection. The genotype of 17 overlapping loci between AGCU Y30 assay and Yfiler was completely identical. (XLSX 156 kb)

Supplementary Table 5

Reproducibility of AGCU Y30 assay between different laboratories. Two male Control DNA and 15 male case samples were detected by three independent laboratories at different time. Each of the same sample was obtained the sample genotyping result from three laboratories. MCD1: Male Control DNA 9948, MCD2: Male Control DNA 007, L1: Laboratory One, L2: Laboratory Two, L3: Laboratory Three, S1-S15: Represents each one of 15 case samples respectively. (XLSX 17 kb)

Supplementary Table 6

The standard deviation in the size value for each allele of AGCU Y30 assay Allelic Ladder. Twenty-four AGCU Y30 assay Allelic Ladder (1 μL of each) were electrophoresed on a 3500xl Genetic Analyzer using a 2 kV, 8 s injection. The minimum and maximum standard deviation was 0.0240 nt at allele 21 of DYS448 and 0.0842 nt at allele 25 of DYS635, respectively. (XLSX 15 kb)

Supplementary Table 7

Reaction volume analysis. Purified DNA (Male Control DNA 9948) and male blood/buccal samples on FTA™ cards were amplified with various reaction volumes. 1 μL of amplification product was electrophoresed on a 3500xl Genetic Analyzer using a 2 kV, 8 s injection. (DOCX 16 kb)

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Zhou, Y., Xie, T., Guo, Y. et al. The validation study of a novel assay with 30 slow and moderate mutation Y-STR markers for criminal investigation and database applications. Int J Legal Med 134, 491–499 (2020). https://doi.org/10.1007/s00414-019-02037-w

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