Abstract
The SureID®S6 system used a lyophilized pellet as the amplification reagent to enable multiplexing of sex-determining marker Amelogenin, 21 autosomal short tandem repeats (STRs), and one Y-STR. To assess the performance, reliability, and limitation of the dry amplification system, the validation studies including PCR condition, reproducibility, sizing and precision, analytical threshold calculation, sensitivity and stochastic threshold calculation, species specificity, stability, mixture, case sample, and population and concordance were conducted according to the Scientific Working Group on DNA Analysis Methods (SWGDAM) Validation Guidelines. Experimental data suggested that the optimal range of total input DNA was from 125 to 500 pg; the appropriate analytical threshold was 80 relative fluorescence units (RFUs) while the stochastic threshold was 260 RFUs; for the stability studies, SureID®S6 system could resist against less than 500 μmol/L of hematin, 100 ng/μl of humic acid, 4 mM of indigotin, 800 mM of tannic acid, and 800 mM of calcium ion. Population and concordance studies using 500 unrelated individuals showed that the combined probability of discrimination (CPD) and cumulative probability of exclusion (CPE) values were 0.999999999999 and 0.999999998416, respectively. The genotypes for the same sample were concordant with the previously validated HUAXIA™ Platinum kit. The validation results demonstrated that the SureID®S6 system could be used for forensic applifications.
Highlights
The amplicons for Amelogenin, D8S1179, D21S11, D18S51, and D2S1338 were in the blue channel, and FAM fluorescence material was used for labeling; the amplified products for D2S441, D5S818, D7S820, D6S1043, and Penta D in the green channel were labeled with HEX; D3S1358, TH01, D19S433, D12S391, and DYS391 were labeled with TAMRA and displayed in yellow; the red channel including TPOX, D16S539, D13S317, and FGA were labeled with ROX; the purple channel included VIG fluoresceinlabeled short tandem repeats (STRs) amplicons for CSF1PO, vWA, D1S1656, and Penta E loci (Supplementary Table S1; Supplementary Figure S1); Internal lane standard (ILS), which contained standard DNA fragments of different sizes, displayed orange fluorescence by labeling SIZE-500(S)
At the optimal cycle number 2 + 27, the profiles led to a maximum number of alleles displaying heterozygous peak heights between 3,000 and 12,000 relative fluorescence units (RFUs) and the minimal occurrence of off-scale peaks
The results showed that the genotyping accuracy of STR profiles was not affected by volume fluctuations in this range
Summary
PCR is widely used to amplify molecular markers, such as short tandem repeat (STR), single nucleotide polymorphism (SNP), insertion-deletion (InDel) and other molecular markers from forensic samples to solve problems, such as paternity testing, individual identification (Hammond et al, 1994), ancestry inference (Guo et al, 2020), age estimation (Marquez-Ruiz et al, 2020), body fluids identification (Ingold et al, 2020), phenotypic inference (Chaitanya et al, 2018), complicated kinship inference (Kling and Tillmar, 2019), and post-mortem interval estimation (Tu et al, 2018). Since repeated freezing and thawing may decrease the enzyme activity, and the operation may . Validation of S6 System increase the possibility of sporadic contamination, these reagents are usually divided into aliquots before storage (Kwok and Higuchi, 1989). (S6), which aimed to simplify PCR procedures by using a lyophilized amplification reagent. The dried reagent mixture of the system is packaged in a separate reaction tube and does not need to be divided into aliquots again. Working with the new system, operators will no longer need to prepare PCR premixtures, and the incidence of sporadic contamination (de Lomas et al, 1992) will decrease at the same time. Apart from the above, advantages of lyophilized reagents include a longer shelf life, lower storage conditions, and ease of transportation
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