Analysis of mock-degraded and mixed DNA samples using the ForenSeq™ MainstAY kit
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Abstract
For more than two decades, the polymerase chain reaction (PCR) amplification of short tandem repeat (STR) polymorphisms followed by detection with capillary electrophoresis (CE) has been the standard method in forensic deoxyribonucleic acid (DNA) analysis. While direct PCR to CE is a relatively inexpensive and highly efficient approach, it is limited in resolution, throughput, and multiplexing ability. The advent of massively parallel sequencing (MPS) offers the potential of overcoming limitations of CE-based approaches due to the increased overall discrimination power, multiplexing capacity of various forensically-relevant markers and number of samples, and the ability to reveal true sequence variation. This offers forensic practitioners the potential to solve the challenges faced by PCR-CE when analyzing forensically challenging samples, such as degraded and mixed DNA. The Verogen ForenSeq™ MainstAY Kit is a MPS library preparation method with a streamlined workflow that allows for the targeted sequencing of 53 standard STR loci on the Illumina MiSeq™ FGx Sequencing System.
This study evaluated the repeatability and performance of the Verogen ForenSeq™ MainstAY Kit in analyzing mock-degraded and mixed DNA samples. The MainstAY Kit produced reliable and repeatable results across replicates. When compared to the GlobalFiler™ Kit for mock-degraded samples, the MainstAY Kit outperformed the standard method with more consistent intralocus balance and signal intensity, along with less dropout observed with increasing degradation. Complete and partial Combined DNA Index System (CODIS) STR profiles were generated using MPS across a wide range of degradation severity, with an 86% complete single-source profile obtained for the most degraded sample (degradation index: 2041.6). The MainstAY Kit successfully detected minor alleles for low level contributor mixtures as low as 6% minor contribution. This study proves the viability of the ForenSeq MainstAY Kit as an alternative solution to conventional fragment analysis, and may be useful to laboratories assessing MPS technologies.