Next-Generation Sequencing: ForenSeq™ DNA Signature Prep Kit with the Illumina MiSeq FGx.

Sequencing forensic DNA samples that are amplified and prepared with the ForenSeq™ DNA Signature Prep Kit allows for the simultaneous targeting of forensically relevant STR and SNP markers. The MiSeq™ FGx system allows massively parallel sequencing of these markers in a single analysis. The library preparation targets autosomal, Y-, and X-STRs, as well as identity SNPs. The kit can also be used to generate investigative information regarding the DNA contributor by analyzing phenotypic SNPs to predict hair color, eye color, and ancestry SNPs.Through two rounds of amplification, all loci are amplified and tagged with individualizing barcodes for sequencing capture and identification. Using bead-based technology, the libraries are purified by the removal of left-over amplification reagents and then normalized to ensure equal representation of all samples during sequencing. The individual libraries are then pooled for insertion into the MiSeq FGx. The pooled libraries are then added to a pre-packaged cartridge that contains all reagents necessary for optimal sequencing. Libraries are captured on a flow cell and undergo bridge amplification for the generation of individual clusters. Sequencing of each cluster is performed using a Sequence-By-Synthesis technology. The following chapter describes the methodology and process of library preparation of samples using the ForenSeq™ DNA Signature Prep Kit Primer Set A and B. Once completed, the chapter then focuses on the setup of a sequencing run on the MiSeq FGx and the sequencing methodology employed by the instrument.