Dataset Information

Developmental validation of Applied Biosystems YFiler Platinum Casework PCR Amplification Kit.

ABSTRACT: The YFiler Platinum Casework PCR Amplification Kit is a 6-dye multiplex assay that simultaneously amplifies a set of 38 male-specific, Y-chromosome Short Tandem Repeat (YSTR) markers (DYS576, DYS389I, DYS635, DYS389II, DYS627, DYS549, DYS593, DYS645, DYS460, DYS458, DYS19, YGATAH4, DYS448, DYS391, DYS557, DYS522, DYS456, DYS390, DYS438, DYS392, DYS518, DYS444, DYS533, DYS570, DYS437, DYS385, DYS449, DYS643, DYS596, DYS393, DYS439, DYS481, DYF387S1, DYS527, DYS447), three insertion/deletion polymorphic markers (Yindels: rs771783753, rs759551978, rs199815934), and an internal quality control (IQC) system. When compared to the YFiler Platinum PCR Amplification kit for database samples, YFiler Platinum Casework kit was developed to include an improved Primer Mix incorporating a brighter TED dye, an updated internal quality control system, better resolution of large DNA fragments in Applied Biosystems POP-4 Polymer, and reduced female DNA cross-reactivity. Here, we report the results of the developmental validation study which followed the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines and includes data for PCR-based studies, sensitivity, species specificity, stability, precision, reproducibility and repeatability, population concordance, stutter, DNA mixtures, and performance on mock casework samples. The results validate the multiplex design as well as demonstrate the kit's robustness, reliability, and suitability as an assay for human identification with casework DNA samples.

SUBMITTER: Lee S

PROVIDER: S-EPMC10477233 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Developmental validation of Applied Biosystems YFiler Platinum Casework PCR Amplification Kit.

Lee Sumin S McAnany Peterjon K PK Chang Chien Wei CW Norona Wilma W Short Marc L ML Mulero Julio J JJ Zhong Chang C

Scientific reports 20230904 1

The YFiler Platinum Casework PCR Amplification Kit is a 6-dye multiplex assay that simultaneously amplifies a set of 38 male-specific, Y-chromosome Short Tandem Repeat (YSTR) markers (DYS576, DYS389I, DYS635, DYS389II, DYS627, DYS549, DYS593, DYS645, DYS460, DYS458, DYS19, YGATAH4, DYS448, DYS391, DYS557, DYS522, DYS456, DYS390, DYS438, DYS392, DYS518, DYS444, DYS533, DYS570, DYS437, DYS385, DYS449, DYS643, DYS596, DYS393, DYS439, DYS481, DYF387S1, DYS527, DYS447), three insertion/deletion polym ...[more]

PMID: 37666878

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Project description:BACKGROUND: WGA (Whole Genome Amplification) in forensic genetics can eliminate the technical limitations arising from low amounts of genomic DNA (gDNA). However, it has not been used to date because any amplification bias generated may complicate the interpretation of results. Our aim in this paper was to assess the applicability of MDA to forensic SNP genotyping by performing a comparative analysis of genomic and amplified DNA samples. A 26-SNPs TaqMan panel specifically designed for low copy number (LCN) and/or severely degraded genomic DNA was typed on 100 genomic as well as amplified DNA samples. RESULTS: Aliquots containing 1, 0.1 and 0.01 ng each of 100 DNA samples were typed for a 26-SNPs panel. Similar aliquots of the same DNA samples underwent multiple displacement amplification (MDA) before being typed for the same panel. Genomic DNA samples showed 0% PCR failure rate for all three dilutions, whilst the PCR failure rate of the amplified DNA samples was 0% for the 1 ng and 0.1 ng dilutions and 0.077% for the 0.01 ng dilution. The genotyping results of both the amplified and genomic DNA samples were also compared with reference genotypes of the same samples obtained by direct sequencing. The genomic DNA samples showed genotype concordance rates of 100% for all three dilutions while the concordance rates of the amplified DNA samples were 100% for the 1 ng and 0.1 ng dilutions and 99.923% for the 0.01 ng dilution. Moreover, ten artificially-degraded DNA samples, which gave no results when analyzed by current forensic methods, were also amplified by MDA and genotyped with 100% concordance. CONCLUSION: We investigated the suitability of MDA material for forensic SNP typing. Comparative analysis of amplified and genomic DNA samples showed that a large number of SNPs could be accurately typed starting from just 0.01 ng of template. We found that the MDA genotyping call and accuracy rates were only slightly lower than those for genomic DNA. Indeed, when 10 pg of input DNA was used in MDA, we obtained 99.923% concordance, indicating a genotyping error rate of 1/1299 (7.7 x 10(-4)). This is quite similar to the genotyping error rate of STRs used in current forensic analysis. Such efficiency and accuracy of SNP typing of amplified DNA suggest that MDA can also generate large amounts of genome-equivalent DNA from a minimal amount of input DNA. These results show for the first time that MDA material is suitable for SNP-based forensic protocols and in general when samples fail to give interpretable STR results.

Project description:BackgroundDNA microarray technology provides a powerful tool for characterizing gene expression on a genome scale. While the technology has been widely used in discovery-based medical and basic biological research, its direct application in clinical practice and regulatory decision-making has been questioned. A few key issues, including the reproducibility, reliability, compatibility and standardization of microarray analysis and results, must be critically addressed before any routine usage of microarrays in clinical laboratory and regulated areas can occur. In this study we investigate some of these issues for the Applied Biosystems Human Genome Survey Microarrays.ResultsWe analyzed the gene expression profiles of two samples: brain and universal human reference (UHR), a mixture of RNAs from 10 cancer cell lines, using the Applied Biosystems Human Genome Survey Microarrays. Five technical replicates in three different sites were performed on the same total RNA samples according to manufacturer's standard protocols. Five different methods, quantile, median, scale, VSN and cyclic loess were used to normalize AB microarray data within each site. 1,000 genes spanning a wide dynamic range in gene expression levels were selected for real-time PCR validation. Using the TaqMan assays data set as the reference set, the performance of the five normalization methods was evaluated focusing on the following criteria: (1) Sensitivity and reproducibility in detection of expression; (2) Fold change correlation with real-time PCR data; (3) Sensitivity and specificity in detection of differential expression; (4) Reproducibility of differentially expressed gene lists.ConclusionOur results showed a high level of concordance between these normalization methods. This is true, regardless of whether signal, detection, variation, fold change measurements and reproducibility were interrogated. Furthermore, we used TaqMan assays as a reference, to generate TPR and FDR plots for the various normalization methods across the assay range. Little impact is observed on the TP and FP rates in detection of differentially expressed genes. Additionally, little effect was observed by the various normalization methods on the statistical approaches analyzed which indicates a certain robustness of the analysis methods currently in use in the field, particularly when used in conjunction with the Applied Biosystems Gene Expression System.

Dataset Information

Developmental validation of Applied Biosystems YFiler Platinum Casework PCR Amplification Kit.

Publications

Developmental validation of Applied Biosystems YFiler Platinum Casework PCR Amplification Kit.

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