Verogen ForenSeq mtDNA Whole Genome Kit

The ForenSeq mtDNA Whole Genome Kit offers a convenient and cost-effective library prep for sequencing the whole mitochondrial genome (mtGenome) with minimal DNA input and exceptional data recovery. Leveraging proven ForenSeq chemistry, the kit produces expanded data sets that promote population studies or provide additional resolution for severely degraded samples. Moreover, this solution has the lowest list price of any commercial whole genome assay. Multiplexing capability further boosts operational efficiency without increases to costs or time.

The ForenSeq mtDNA Whole Genome Kit features the highest resolution for mtGenome sequencing in the Verogen library prep portfolio. Tiled amplicons, fit-for-purpose buffers, and a thoughtfully designed protocol enable support for the lower end of DNA input amounts, various sample types, and a broad range of forensic and database applications, including missing persons, disaster victim identification (DVI), and population studies.

• High sensitivity across a range of gDNA inputs
• Enhanced recovery of low input samples
• Robust resistance to common inhibitors

The protocol includes an optional second purification to further improve the performance of low-level and complex samples. The figure “High coverage across a range of input DNA amounts” presents a comparison of coverage results for libraries with and without the second purification. Compared to a single purification, two purifications show progressive coverage advancements, culminating with 90% improvement at 2 pg (see figure “Read count improvement with a second purification”). Another challenge of forensic sample analysis is the frequent presence of PCR inhibitors that can compromise library prep. When varying amounts of three inhibitors common in forensic mtDNA samples were added to 100 pg of Control DNA HL60, results shown in the figure “Buffers highly resistant to relevant PCR inhibitors” demonstrated high resistance with consistent coverage across all inputs.

The targeted PCR-based assay deploys small amplicons sourced from the latest mtDNA databases for enhanced variant detection. A tiled primer approach overlaps the amplicons to prevent the sequence gaps that can lead to data loss, particularly in degraded samples. Complementing this tiled primer design, an enhanced buffer system delivers exceptional resistance to calcium, humic acid, and other relevant PCR inhibitors.

This user-friendly, automation-compatible solution provides a common workflow for a variety of study designs spanning multiple applications. The protocol includes two normalization methods to tailor library prep for a broad range of input material, from high-quality genomic DNA (gDNA) through low-level and complex samples. A low input requirement expands possibilities for the most challenging samples. For efficient use of the reagent kit and accurate read assignment, index adapters provided in the library prep kit allow pooling and sequencing of up to 16 samples in one run. The tiled amplicon design of the whole genome kit is suited for analysis of the entire 16,569 bp human mtGenome. The assay includes reagents to amplify approximately 500 small amplicons and cover the entire genome in two PCR reactions.

Mitochondrial DNA is a powerful alternative source of information when nuclear DNA is compromised or absent. Used for rootless hairs, teeth, bones, and other challenging sources, mtDNA has become routine for missing persons and disaster victim identification efforts. Previously, quality and scaling issues associated with traditional Sanger sequencing methods have confined mtDNA to a limited role in a few specialty centers. Verogen mtDNA workflows offer a unique combination of performance, efficiency, and ease of use so that any lab, of any size, can transition mtDNA analysis from Sanger sequencing to NGS, or improve results obtained from other NGS technologies.

For rapid review of mtDNA data, the ForenSeq mtDNA Whole Genome Kit pairs seamlessly with the ForenSeq mtDNA Analysis Module in Universal Analysis Software (UAS). UAS provides an NGS platform for analyzing and managing forensic genomic data, simplifying complex bioinformatics. The software analyzes sequencing output for the whole mitochondrial genome and provides results less than one hour after run completion. Laboratories can review summarized or detailed results and compare up to nine samples on a clear and interactive user interface. Reports are compatible with multiple DNA databases.

We offer superior support across the entire workflow, from library prep to sequencing to analysis. Our experienced team provides comprehensive service coverage for your equipment and software, validation plans, and implementation guidance so you can quickly operationalize your workflows with ease.