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Performance of a next generation sequencing SNP assay on degraded DNA



Katherine Gettings, Kevin M. Kiesler, Peter Vallone


Forensic DNA casework samples are often of insufficient quantity or quality to generate full profiles by conventional DNA typing methods. Amplification of STR loci is inherently limited in samples containing degraded DNA, as the cumulative size of repeat regions, primer binding regions, and flanking sequence is often larger than the template. Additionally, traditional capillary electrophoresis (CE) assay design further inherently limits shortening amplicons because the markers must be separated by size. Non-traditional markers, such as single nucleotide polymorphisms (SNPs) and insertion deletion polymorphisms (InDels), may yield more information from challenging samples due to their smaller amplicon size. In this study, the performance of a next generation sequencing (NGS) SNP assay as well as CE-based STR, mini-STR, and InDel assays, on a series of fragmented, size-selected samples was evaluated. Information obtained from NGS SNP assays exhibited higher overall inverse random match probability (1/RMP) values compared to the CE-based typing assays, with a particular benefit seen when fragment sizes are ≤ 150 bp. Despite the fact that InDels and mini-STRs show similar percentages of loci with reportable alleles at this level of degradation, the relatively lower number of loci in these assays compared to the NGS SNP assay results in the latter showing at least nine orders of magnitude higher 1/RMP values. In addition, the NGS SNP assay, two CE-based STR assays, and an InDel assay were tested using a dilution series consisting of 0.5 ng, 0.1 ng, and 0.05 ng non-degraded DNA. All tested assays showed similar percentages of loci with reportable alleles at these levels of input DNA; however, due to the larger number of loci, the NGS SNP assay and the larger of the two tested CE-based STR assays both resulted in considerably higher 1/RMP values than the other assays. These results indicate the potential advantage of NGS SNP assays, particularly for forensic analysi
Forensic Science International: Genetics


forensic DNA, SNP, next generation sequencing, degraded DNA


Gettings, K. , Kiesler, K. and Vallone, P. (2015), Performance of a next generation sequencing SNP assay on degraded DNA, Forensic Science International: Genetics, [online], (Accessed May 29, 2024)


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Created May 26, 2015, Updated November 10, 2018