Human DNA profiling using PCR at polymorphic short tandem repeat (STR) loci followed by capillary electrophoresis (CE) size separation and length-based allele typing has been the standard in the forensic community for over 20 years. Over the last decade, Next-Generation Sequencing (NGS) matured rapidly, bringing modern advantages to forensic DNA analysis. The MiSeq FGx™ Forensic Genomics System, comprised of the ForenSeq™ DNA Signature Prep Kit, MiSeq FGx™ Reagent Kit, MiSeq FGx™ instrument and ForenSeq™ Universal Analysis Software, uses PCR to simultaneously amplify up to 231 forensic loci in a single multiplex reaction. Targeted loci include Amelogenin, 27 common, forensic autosomal STRs, 24 Y-STRs, 7 X-STRs and three classes of single nucleotide polymorphisms (SNPs). The ForenSeq™ kit includes two primer sets: Amelogenin, 58 STRs and 94 identity informative SNPs (iiSNPs) are amplified using DNA Primer Set A (DPMA; 153 loci); if a laboratory chooses to generate investigative leads using DNA Primer Set B, amplification is targeted to the 153 loci in DPMA plus 22 phenotypic informative (piSNPs) and 56 biogeographical ancestry SNPs (aiSNPs). High-resolution genotypes, including detection of intra-STR sequence variants, are semi-automatically generated with the ForenSeq™ software. This system was subjected to developmental validation studies according to the 2012 Revised SWGDAM Validation Guidelines. A two-step PCR first amplifies the target forensic STR and SNP loci (PCR1); unique, sample-specific indexed adapters or "barcodes" are attached in PCR2. Approximately 1736 ForenSeq™ reactions were analyzed. Studies include DNA substrate testing (cotton swabs, FTA cards, filter paper), species studies from a range of nonhuman organisms, DNA input sensitivity studies from 1ng down to 7.8pg, two-person human DNA mixture testing with three genotype combinations, stability analysis of partially degraded DNA, and effects of five commonly encountered PCR inhibitors. Calculations from ForenSeq™ STR and SNP repeatability and reproducibility studies (1ng template) indicate 100.0% accuracy of the MiSeq FGx™ System in allele calling relative to CE for STRs (1260 samples), and >99.1% accuracy relative to bead array typing for SNPs (1260 samples for iiSNPs, 310 samples for aiSNPs and piSNPs), with >99.0% and >97.8% precision, respectively. Call rates of >99.0% were observed for all STRs and SNPs amplified with both ForenSeq™ primer mixes. Limitations of the MiSeq FGx™ System are discussed. Results described here demonstrate that the MiSeq FGx™ System meets forensic DNA quality assurance guidelines with robust, reliable, and reproducible performance on samples of various quantities and qualities.
Automated fluorescence analysis of polymerase chain reaction (PCR)-amplified short tandem repeat (STR) systems by capillary electrophoresis (CE) is becoming an established tool both in forensic casework and in the implementation of both state and national convicted offender DNA databases. A new capillary electrophoresis instrument, the ABI Prism 310 Genetic Analyzer, along with the Performance Optimized Polymer 4 (POP-4) provides an automated and precise method for simultaneously analyzing ten fluorescently labeled STR loci from a single PCR amplification kit, which provides a power of discrimination of approximately one in five billion from a single PCR amplification. Data are presented on sizing precision, sizing accuracy, and resolution for the STR loci in the AmpFlSTR Profiler kit. Sizing accuracy is highly dependent on the electrophoresis system, and therefore the reporting of alleles based on the nucleotide size obtained from an electrophoresis system is not recommended for forensic work. The precision of the 310 capillary electrophoresis system, coupled with software developed for automated genotyping of alleles based on the use of an allelic ladder, allows for accurate genotyping of STR loci. Sizing precision of < or = 0.16 nucleotide standard deviation was obtained with this system, thus allowing for accurate genotyping of length variants that differ in length by a single nucleotide.
The 944 individuals of the CEPH human genome diversity panel (HGDP-CEPH), a standard sample set of 51 globally distributed populations, were sequenced using the Illumina ForenSeq™ DNA Signature Prep Kit. The ForenSeq™ system is a single multiplex for the MiSeq/FGx™ massively parallel sequencing instrument, comprising: amelogenin, 27 autosomal STRs, 24 Y-STRs, 7 X-STRs, and 94 SNPforID+Kiddlab autosomal ID-SNPs (plus optionally detected ancestry and phenotyping SNP sets). We report in detail the patterns of sequence variation observed in the repeat regions of the 58 forensic STR loci typed by the ForenSeq™ system. Sequence alleles were characterized and repeat region structures annotated by aligning the ForenSeq™ sequence output to the latest GRCh38 human reference sequence, necessitating the reversal and re-alignment of STR allele sequences reported by the Forenseq™ system in 20 of 58 STRs (plus the reverse alleles in two Y-STRs with duplicated-inverted repeat regions). Individual population sample sizes of the HGDP-CEPH panel do not allow reliable inferences to be made about levels of genetic variability in low frequency STR alleles-where particular sequence variants are found in only a few individuals; but we assessed the occurrence of both population-specific sequence variants and singleton observations; finding each of these in a sizeable proportion of HGDP-CEPH samples, with consequences for planning the co-ordinated compilation of sequence variation on a much larger scale than was required before by forensic laboratories now adopting massively parallel sequencing.
Laboratory procedures used in short tandem repeat (STR) analysis were subjected to various scenarios that assessed reliability and identified potential limitations. These validation studies were designed as recommended by the Technical Working Group on DNA Analysis Methods (TWGDAM) and the DNA Advisory Board (DAB) (17,18). Various DNA samples were amplified by the polymerase chain reaction (PCR) using AmpFᐉSTR™ PCR Amplification Kits (i.e., AmpFᐉSTR Green I, Profiler™, Profiler Plus™, and COfiler™ kits), detected with ABI Prism instrumentation, and analyzed using GeneScan and Genotyper software. Data acquired in these studies reinforced an existing body of knowledge and expertise regarding application and interpretation of STR typing in the forensic science community. Consistent STR genotypes were detected in various body tissues and fluids. Inter-laboratory comparisons produced concordant genotype results. Quantitative interpretational aids for DNA mixtures were characterized. Ability of the typing systems to type potentially compromised samples reliably was evaluated. Nonprobative case evidentiary DNA was successfully amplified, genotyped, and interpreted. Potential limitations or cautionary factors in the interpretation of minimal fluorescence intensity were demonstrated. Differential amplification between loci was observed when PCR was inhibited; preferential amplification typically was not. Single AmpFᐉSTR locus amplification did not offer consistent benefit over AmpFᐉSTR multiplexing, even in cases of DNA degradation or PCR inhibition. During rigorous evaluation, AmpFᐉSTR PCR Amplification Kits reproducibly yielded sensitive and locusspecific results, as required in routine forensic analyses.
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