Current forensic DNA analysis utilizes capillary electrophoresis (CE) to separate short tandem repeat (STR) fragments based on their length. Next generation sequencing (NGS) is the next evolution of forensic DNA profiling, and though dedicated forensics protocols are still fairly new, it is only a matter of time before NGS becomes the new standard for forensic DNA profiling. Stutter has been a problem ever since forensic STR testing was first implemented. The slipped strand mispairing model is the proposed mechanism for how stutter occurs, and it appears to be an intrinsic part of the polymerase chain reaction (PCR). Samples that were run via the ForenSeq™ DNA Signature Prep Kit were amplified using the DNA Primer Mix A (DPMA) and then sequenced on a MiSeq FGx™ Forensics Genomics System. Samples were also amplified using the GlobalFiler™ PCR Amplification kit and fragment separation was done via capillary electrophoresis. Stutter ratios were calculated by dividing the read count /relative fluorescence unit of the stutter allele by the read count / relative fluorescence unit of the parent allele. Stutter ratio comparisons were made between the ForenSeq™ and GlobalFiler™ kits as well as between the parents and the children in the family pedigree, though only samples tested using the ForenSeq™ kit were used. Comparison of overall stutter ratios revealed that the ForenSeq™ kit produced higher stutter across all STR loci, except for D13S317 and D2S441, when compared to the GlobalFiler™ kit. The different chemistries between the two kits, potential usage of different polymerases, and the fact that the ForenSeq™ kit requires two rounds of amplification can serve as likely explanations for this difference. There was also quite a bit of variability observed for the stutter ratios between loci in the samples run using the ForenSeq™ kit. Possible explanations for this could be that the cluster generation step could produce more clusters for some stutter products over others. Comparison of the stutter ratios for the pedigree obtained from the Coriell Institute revealed no differences between the parents and the respective alleles inherited by the children when tested with the ForenSeq™ kit. Some loci showed a difference between the parent and children, but that could simply be due to the sample size. The utilization of NGS for STR testing can result in two alleles of the same length but different sequences, called isoalleles. Analysis of isoalleles present at D21S11 in the children samples from the Coriell Institute pedigree showed that the isoalleles had different mean stutter ratios. The results open the possibility of potentially utilizing sequence-specific stutter filters in the ForenSeq™ Universal Analysis Software. The model of the longest uninterrupted stretch (LUS) has been around for some time, though recently the block length of missing motif (BLMM) has been proposed as a better predictor for stutter ratios. The results of stutter ratio analysis at D21S11 show that as the length of the BLMM increases, so too does the stutter ratio.

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.