Improving resolution of mixtures by DNA sequencing using the Illumina MiSeq FGx system
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The use of short tandem repeats (STRs) for genotyping forensic case samples has long been an effective tool for human identification. However, interpretation of forensic STR mixture samples can be difficult and any additional information to aid in this process can be invaluable. Allele overlap and stutter during PCR can cause drop out of the minor contributor’s alleles and result in incorrect allele calling. The Scientific Working Group on DNA Analysis Methods (SWGDAM) provides a list of guidelines on how to interpret DNA typing results from forensic STRs and mixtures, but there is still a significant variation in the interpretation of mixture samples between analysts in the same laboratory and between laboratories. The Illumina MiSeq Forensic GenomicsTM system (Illumina Inc., San Diego, CA) is a massively parallel sequencing instrument that was developed specifically for the use in forensic DNA typing and which could provide sequence variations among on mixture samples. The ForenSeqTM DNA Signature Prep Kit is a kit that can be used with the MiSeq FGxTM platform. The DNA Primer Mix A (DPMA) included in the ForenSeqTM kit targets 27 autosomal STRs, 24 Y-STRs, 7 X-STRs and 94 identity single nucleotide polymorphisms (SNPs) on up to 32 or 96 samples, depending on the flow cell used. This study compares the STR performance on DNA mixtures of the MiSeq FGxTM and CE and evaluates its reliability and robustness. The MiSeq FGxTM provides data in read count and the CE in relative fluorescence units (RFU), so the two output data cannot be directly compared to one another. Instead, the ratio of two contributors was calculated at three mixture ratios (1:1, 1:4, and 1:9) to use as a mean of comparison. The mean contributor ratios calculated on the MiSeq FGxTM were 1.799, 7.595, and 13.524 for the 1:1, 1:4, and 1:9 mixtures, respectively. This was not significantly different from the CE mean contributor ratios of 1.818, 7.722, and 14.827, respectively. More allele dropouts occurred on the MiSeq FGxTM than the CE at both 1:4 and 1:9 mixture ratios, but sequencing provided the detection of six isoalleles based on sequence variants that could not be discerned by CE. Other studies have shown full profile generation at these ratios, indicating there could have been some issues during library preparation. Further studies should be performed to thoroughly validate the ForenSeqTM process and evaluate the sensitivity of the instrument. Until then, it is recommended that the ForenSeqTM kit and MiSeq FGxTM system be used at close to equal mixture ratios or in tandem with the CE to prevent genotypes miscalling.