Stutter analysis of a family pedigree via massively parallel sequencing utilizing the ForenSeq DNA Signature Prep kit
Porto, Andre C.
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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.
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