Independent development and validation of a novel six‐color fluorescence multiplex panel including 61 diallelic DIPs and 2 miniSTRs for forensic degradation sample

Abstract

Current forensic DNA profiles are obtained based on analyses of PCR product sizes or DNA sequence polymorphisms. Sometimes routine forensic analysis using short tandem repeat (STR) generates unsuccessful DNA testing result if the biological sample encountered is excessively degraded and low-template DNA. Herein, a new six-color fluorescence labeling system, including 59 autosomal diallelic deletion or insertion polymorphisms (DIPs), 2 miniSTRs, 2 Ychromosome DIPs, and 1 Amelogenin gene with the amplicon sizes of less than 200 bp, was self-developed. According to the validation guidelines for DNA analysis methods formulated by the Scientific Working Group on DNA Analysis Methods, the validation studies have also been carried out for the multiplex system. This novel panel possessed the features of strong stability, high sensitivity, and good specificity, which was especially suitable for the forensic degraded and mixed sample detections. The cumulative power of exclusion and cumulative matching probability of the system were 0.9999978 and 9.833E-28, respectively, in Han Chinese in Hunan, China. Moreover, this system will be an effective new tool that can be independently applied to forensic personal identification and paternity testing in the populations from the East Asia region, even from the South Asia, America, and Europe regions. The system can also contribute to Abbreviations: CDX,

Forensic Characteristics and Genetic Background Dissections of Chinese Manchu and Zhuang Groups Using 59 Autosomal Diallelic InDels and 2 miniSTRs

“…Attributed to the construction of the home-made 6-dye multiplex amplification panel [8], which is comprised of 59 autosomal InDels, 2 miniSTRs, 2 Y-InDels and an Amelogenin (AMEL) gene, it is now possible to further improve the effi-cacy of the multiplex panel for individual identification and paternity testing by combining diallelic InDels with shorter amplicons and miniSTRs with higher polymorphisms in a multiplex panel. Moreover, as a result of the addition of two miniSTRs, the 64-plex panel was able to indicate the presence of a mixture sample by observing the presence of the third or more alleles in one miniSTR locus, and the minor component was explicitly detected in those samples with the mixed ratios of 1:9 and 9:1 in the former validation study [7]. Although the forensic applicability of the novel selfdeveloped 64-plex has been preliminarily validated [7], the polymorphic data of various populations from China still need to be investigated by the panel, and to further evaluate the robustness of the panel for the efficacies of individual identification and paternity testing.…”

“…Moreover, as a result of the addition of two miniSTRs, the 64-plex panel was able to indicate the presence of a mixture sample by observing the presence of the third or more alleles in one miniSTR locus, and the minor component was explicitly detected in those samples with the mixed ratios of 1:9 and 9:1 in the former validation study [7]. Although the forensic applicability of the novel selfdeveloped 64-plex has been preliminarily validated [7], the polymorphic data of various populations from China still need to be investigated by the panel, and to further evaluate the robustness of the panel for the efficacies of individual identification and paternity testing.…”

“…Even though most of the diallelic InDel-based different panels could meet the need for individual identification, they still fail to obtain comparable efficacies to that of STRs in paternity testing and are not so instructive when interpreting and deconvoluting DNA mixture [5]. In contrast, by shortening the lengths of the upstream and downstream flanking sequences of the core repetition region in the STR marker, miniSTR has dozens of alleles like traditional common STR and a shorter size of amplicon, resulting in its higher polymorphic information and better forensic applicability in mixture analysis than those of InDel genetic marker [6,7]. However, due to the multi-allelic nature of miniSTR which consists of multiple different repeat motifs with 2-6 bp in length, some loci with more alleles in the commercial min-iSTR kits, such as MiniFiler™ PCR Amplification kit, still are relatively big amplicon sizes of over 200 bp, which limits the number of loci within the same color fluorescence lane, and also potentially leads to DNA typing failure in the highly degraded sample, where allele dropout is more likely to happen in those miniSTRs with bigger amplicons.…”

Insight into forensic efficiency and genetic structure of the Guizhou Dong group via a 64-plex panel

“…The mixture comprised 2 μL Master Mix (HEALTH Gene Technologies, Ningbo, China), 2 μL Primer Mix, and 6 μL deionized water. Next, the PCR cocktail was amplified on the GeneAmp PCR System 9,700 equipment (Thermo Fisher Scientific, Foster City, CA, United States) according to the recommended parameters (Liu et al, 2022b). Thirdly, we extracted 1 μL amplified product and added it into the cocktail including 0.5 μL SIZE-500 (HEALTH Gene Technologies) and 8.5 μL Hi-Di deionized formamide.…”

“…Previously, a novel multiplex amplification system (64-plex) that includes two STRs, 59 InDels, and three sex-determination loci has been developed based on the CE (Liu et al, 2022a). In the following study of developmental validation, Liu et al (2022b) found that the novel panel displayed greater application value in forensic individual identification and kinship testing than the extant InDel and miniSTR panels. In addition, the novel panel showed good tolerance to common inhibitors, strong species specificity, and good applicability for case-type samples.…”