A comparison of the Illumina MiSeq FGX™ System against capillary electrophoresis in the analysis of two-person mixtures
McEvoy, David Patrick
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The following is a comparison study of the Globalfiler™ PCR Amplification Kit analyzed on an ABI 3130 Genetic Analyzer Capillary Electrophoresis (CE) versus the ForenSeq™ DNA Signature Prep Kit analyzed on the MiSeq FGx™ System. The MiSeq FGx™ System measures results by Allele Read Count (ARC), while the CE measures results as Relative Fluorescent Units (RFU). Mixture samples were prepared in ratios of 1:1, 1:4, and 1:10 in replicates of four using a female major contributor and a male minor contributor, intended to represent some commonly seen mixture samples ratios in forensic cases . Both systems performed equally well for the 1:1 mixture while the MiSeq FGx™ System had improved accuracy and precision for the 1:4 mixture compared to CE (4.033 + 1.506 ARC and 4.678 + 2.093 RFU, respectively). The MiSeq FGx™ System showed increased variation in the 1:10 mixture compared to CE (10.347 + 5.184 ARC and 9.311 + 3.363 RFU, respectively). Over the four replicates, the MiSeq FGx™ System had a total of 15 out of 528 possible alleles (2.84%) dropout compared to a total of 13 out of 384 possible alleles (3.39%) dropout on CE. The additional loci analyzed by the MiSeq FGx™ System results in a lower percentage of alleles lost due to dropout compared to CE. Isoalleles in sequence data may reveal the presence of minor contributor alleles that would otherwise be masked by the major contributor in length-based STR analysis. The presence of isoalleles are most helpful in mixture ratios greater than 1:1, where it is easier to assign alleles to a specific contributor. In certain cases, deconvolution of loci with shared alleles may not be improved by sequencing if intra-contributor isoalleles are present. Unless using known reference profiles, it is difficult to accurately assign alleles to contributors when intra-contributor isoalleles are present. Additionally, the sequencing data from the MiSeq FGx™ System provided information to aid the separation of stutter from true alleles. Previous studies report a significant increase in the amount of alleles present at some loci due to differences by nucleotide sequence, which may improve the discriminating power of those loci [31,52,53,54]. With all its potential, there is still much room for sequencing technology to improve before it becomes a standard analysis method in forensic laboratories.