Verogen ForenSeq Imagen Kit

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.

Includes the required reagents to process up to 96 samples simultaneously for phenotypic and biogeographical ancestry information to generate comprehensive DNA profiles

Features

• Evaluation of phenotypic SNPs with or without biogeographical ancestry
• Workflow tailored to integrate in operational forensic laboratories
• Interoperable and scalable with preconfigured reports for upload to third party tools

Performance

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

Principle

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).

Procedure

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.

Applications

Forensic DNA Phenotyping (FDP) can predict externally visible characteristics from DNA traces at a crime scene. FDP analysis can help focus police investigations with information on an unknown perpetrator when standard DNA profiling fails to inform on identification.

The simple Verogen ForenSeq Imagen Kit plate-based assay is part of an integrated FDP workflow that sequences libraries on the MiSeq FGx Sequencing System and analyzes data in Universal Analysis Software (UAS). Seamless report integration with 3rd party software such as HIrisPlex-S allows users to access enhanced lead generation insights.

Software

Data generated by the ForenSeq Imagen kit is optimized for analysis using the Universal Analysis Software. Two fit-for-purpose analysis methods enable labs to analyze data from the DPME or DPMF (Geo) primer mixes separately. The UAS generates SNP reports that can be uploaded to the HIrisPlex-S web application for predictions powered by solutions described in multiple peer-reviewed publications.

To allow easy interpretation of complex statistical information, reports from the HIrisPlex-S web application can be parsed by the Imagen-HIrisPlex-S Analysis Tool (I-HAT) application on the UAS data server. The I-HAT macro generates human-readable reports that summarize the predictions and probabilities associated with typed SNPs.

Automation of the lab workflow allows labs to onboard the entire ForenSeq Imagen workflow from lab to report with confidence. Robust out-of-the-box thresholds and settings allow easy validation and implementation. Rich data visualization capabilities enable labs to quickly gauge the quality of their sequencing run and SNP calls. Extensive filtering and sorting capabilities enable manual review of markers that require inspection.

Documents

• ForenSeq Imagen Kit
• ForenSeq Imagen Kit with the ForenSeq Enhanced PCR1 Buffer System
• ForenSeq Imagen Kit Handbook for PrepStation
• ForenSeq Imagen Kit Reference Guide
• ForenSeq Imagen Kit with the Enhanced PCR1 Buffer System Reference Guide
• Universal Analysis Software Imagen Module V2 Reference Guide # VD2022014 Rev. A August 2022
• A new horizon of human identification possibilities with NGS
• Reimagining Human Identification

Ordering Information

Cat. no.	Product	Contents
V16000189	Verogen ForenSeq Imagen Kit	Includes the required reagents to process up to 96 samples simultaneously for phenotypic and biogeographical ancestry information to generate comprehensive DNA profiles

The Verogen ForenSeq Imagen Kit is intended for molecular biology applications in forensic, human identity, and paternity testing. This product is not intended for diagnosis, prevention, or treatment of a disease.