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WIREs Forensic Sci

A review of the method and validation of the MiSeq FGx™ Forensic Genomics Solution

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Abstract The MiSeq FGx™ Forensic Genomics Solution (MFGS; Verogen Inc.) is a massively parallel sequencing workflow using Illumina sequencing technology. The workflow includes the ForenSeq™ DNA Signature Prep kit, the MiSeq FGx™, and the ForenSeq™ Universal Analysis Software (UAS). This review explores the molecular biology and bioinformatic methods used during the preparation of samples into sequencing libraries, the MiSeq FGx™ sequencing process, and the data analysis. We highlight what happens during each of these steps and how they influence the final results, while identifying limitations and possible improvements. After initial evaluations established its suitability for forensic applications, a number of studies have completed forensic validation studies. We review this work, and find the MFGS has shown to be a reliable and robust technology. The successful validation of the solution has allowed its implementation into forensic casework in a number of jurisdictions. Some questions still remain around the normalization of the sequencing libraries, and the bioinformatic processing and analysis of the sequencing results. In particular, the markers for biogeographical ancestry and externally visible characteristics require further research into their performance, and the ForenSeq™ UAS, in its current form, is limited in its ability to accurately predict these characteristics. We suggest a number of alternative bioinformatic tools that could be used instead. This article is categorized under: Forensic Biology > Forensic DNA Technologies Forensic Biology > Phenotypic Markers Forensic Biology > Ancestry Determination using DNA Methods
The steps involved in sequencing samples using the MiSeq FGx™ Forensic Genomics Solution
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Sequencing by synthesis reaction. Diagrammatic representation of a library fragment, including the sequencing primer binding positions and direction of sequencing by synthesis. The sequencing of read 1 begins with the sequencing primer (Rd1 Seq primer) hybridizing to the 3′ end of the P5 adapter sequence of the forward strand, and read 1 is sequenced by synthesis. The i7 index sequencing is primed by the i7 Seq primer. The library fragment bends and binds to a flowcell attached P5 oligonucleotide to form a bridge, this P5 oligo acts as the sequencing primer for the sequencing of the i5 index read. Following the reverse strand synthesis and denaturing, read 2 is sequenced primed by the read 2 sequencing primer (Rd2 Seq primer)
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Enrich targets. Diagrammatic representation of the adapter incorporation and target enrichment PCR2 reaction. The i5 illumina adapter binds to the Fwd tag of the PCR1 product, this contains the Fwdv tag compliment, the i5 index, and the P5 flowcell olignucleotide compliment. The i7 illumina adapter binds to the rev tag of the PCR1 product, this contains the rev tag compliment, the i7 index, and the P7 flowcell olignucleotide compliment
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Target amplification. Diagrammatic representation of the target amplification PCR1 reaction. The gDNA primer binding sites are labeled Fwd for the forward primer and rev for the reverse primer (gray), these are specific for each target in the DNA primer mix. The 5′ sequence is complementary to the Illumina adapters used in the adapter incorporation and target enrichment step (red and purple labeled Fwd tag and rev tag)
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Forensic Biology > Ancestry Determination using DNA Methods
Forensic Biology > Phenotypic Markers
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