Despite the prevalent utilization of capillary electrophoresis (CE) in the analysis of short tandem repeats (STRs) to generate deoxyribonucleic acid (DNA) profiles for forensic comparisons, the method is not without its inherent drawbacks. Massively parallel sequencing (MPS) is still a relatively novel technology in the forensics field, but contains the capacity to address current challenges faced by the traditional CE approach - such as degraded samples, low template DNA, and artifacts - while also providing additional information such as isoalleles, same-length alleles with sequence variation, and ancestry, mixture, and phenotyping-informative single nucleotide polymorphisms (SNPs).
One of the principal ongoing challenges faced by both technologies is the presence of artifacts such as stutter, a byproduct of slipped strand mispairing during amplification of STRs, which can further complicate interpretation of DNA profiles. Understanding and predicting the behavior of stutter is important in establishing appropriate thresholds to distinguish these artifacts from true alleles. With complex MPS data, new approaches in characterizing stutter have been established such as the BLMM and simplified sequence.
In this study, twenty-one oral samples from individuals belonging to the same family were constructed into libraries containing 58 STR regions and 98 identity SNPs using Verogen’s Forenseq™ DNA Signature Prep Kit and sequenced on the MiSeq FGx™ Forensics Genomics System. Isoallele and stutter sequences were extracted from the data and simplified using the longest uninterrupted stretch (LUS), block length of missing motif (BLMM) and simplified sequence approaches. It was found that the stutter ratio for the 11 isoallele pairs at the D13S317 locus did not follow the linear correlation with increasing LUS. Instead, the isoallele with the higher LUS demonstrated equal or lower stutter ratios. Additionally, three different stutter patterns were identified for the same locus. Based on the provided pedigree, ten different relations were defined and the amount of allele sharing between the individuals in the pedigree was analyzed with and in the absence of isoallelic information to determine its impact on predicting relatedness. It was found that there was an average of 1.3% difference across the ten defined categories when isoalleles were taken into consideration. However, the difference in the percentage of shared alleles was not found to be significant for each of the relations category between the results before and after the consideration of isoallelelic data.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/42211 |
| Date | 01 March 2021 |
| Creators | Wu, Ping Yi |
| Contributors | Brodeur, Amy N. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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