Description
The Verogen ForenSeq Imagen kit is a library preparation kit offering two different primer mixes that allow the user to evaluate phenotypic SNPs with or without biogeographical ancestry. The ForenSeq Imagen workflow is a fully validated DNA-to-data workflow specifically designed for forensic genomics applications. The kit relies on established, peer-reviewed SNP marker sets that have been identified and tested by the forensic community. The Verogen ForenSeq Imagen Kit is an optimized solution for operational forensic labs considering implementing an NGS-based lead generation workflow.
The ForenSeq Imagen Kit offers the following features: A concentrated primer mix for increased sample input volume Simultaneous preparation of up to 96 libraries in one plate; each library is a collection of amplified DNA fragments from one sample Assay designed to maximize DNA profiles with degraded and low-input samples by generating short amplicons with Unique Dual Indices (UDIs) Buffer system to support inhibited forensic samples Two primer mixes to generate insights based on existing and emerging regional regulation DPME – Amplifies SNP targets for the prediction of skin, hair and eye color, also referred to as external visible characteristics (EVC) DPMF (Geo) – Amplifies SNP targets for the prediction of EVCs in DPME and biogeographical ancestry (BGA) markers Both primer sets encompass 41 SNPs for the phenotypic estimations that include 22 SNPs for the estimation of hair color, 6 SNPs for the estimation of eye color, 36 markers for the estimation of skin color, with some SNPs common to more than one phenotype. Both primer sets encompass 14 Y-SNPs to enable robust estimation of biological sex. In addition, 56 SNPs for the estimation of biogeographic ancestry are included in one of the primer sets. Labs have the flexibility to use either of the two primer sets based on the regulatory approaches associated with their geography. Feature Details Sample type Extracted gDNA from a range of common forensic sample types: buccal swab lysate, bones, blood, teeth and FTA card Recommended input for gDNA 32 pg–1 ng per sample Recommended input for FTA card punch 1.2 mm per sample Recommended multiplexing capacity 8–96 libraries per run Number of primer mixes 2: DPME and DPMF (Geo) Primer mix content DPME: 22 hair SNPs, 6 eye color SNPs, 36 skin color SNPs and 14 Y-SNPs (total of 55 SNPs) DPMF (Geo): 22 hair SNPs, 6 eye color SNPs, 36 skin color SNPs, 56 BGA SNPs and 14 Y-SNPs (total of 111 SNPs) Short amplicon detection High concentration of markers <150 bp Total library prep time 7 hours and 15 minutes Hands-on time 1 hour and 30 minutes Total sequencing time ~18 hours with the Verogen MiSeq FGx Reagent Micro Kit
The Verogen ForenSeq Imagen Kit generates dual-indexed libraries for sequencing. Each sample is combined with a primer mix that contains a pair of tagged oligos for each target sequence. PCR cycles link the tags to copies of each target, forming DNA templates consisting of the regions of interest flanked by universal primer binding sequences. The tags are then used to attach index adapters and the resulting library is amplified, purified, and pooled for sequencing. The table provides a summary of the number of markers associated with both primer sets in the Verogen ForenSeq Imagen Kit. SNP category Number of markers Amplicon size range (bp) Included in DNA primer Mix E Mix F Y-SNPs* 14 88–130 Yes Yes Phenotypic SNPs 41 69–227 Yes Yes Hair color 22 92–227 Yes Yes Eye color 6 73–119 Yes Yes Skin color 36 69–213 Yes Yes Biogeographical** ancestry SNPs 56 67–200 No Yes * 4 SNPs overlap across eye, skin and hair color phenotypes; 13 SNPs overlap across hair and skin color phenotypes; and 2 SNPs overlap across eye and skin color phenotypes. ** Only available in the EVC+BGA SNP panel (DPMF-Geo).
The ForenSeq Imagen workflow, comprising the ForenSeq Imagen kit, the MiSeq FGx Sequencing System with the MiSeq FGx Reagent Micro kit, and the ForenSeq Imagen analysis methods in the Universal Analysis Software (UAS), is a fully validated DNA-to-data workflow specifically designed for forensic genomics applications (see figure Verogen ForenSeq Imagen Kit workflow). Prepare the ForenSeq Imagen assay with DPME for External Visible Characteristics (EVC) and Appearance SNPs or DPMF (Geo) for EVC and Biogeographical Ancestry (BGA) SNPs. Sequence with the MiSeq FGx Sequencing System using the MiSeq FGx Reagent Micro Kit. Analyze with dedicated analysis modules preconfigured for Imagen and Imagen Geo analysis methods. Batch-download multiple report types from Imagen and Imagen Geo. Phenotype analysis reports are compatible with the HIrisPlex-S web application. Upload the phenotype report to the HIrisPlex-S web application for estimations. Generate your reports. The data is reformatted in accessible form with the I-HAT macro tool hosted on the Verogen Data Analysis Server.
