Qiagen, V16000086, 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. Feature Details DNA input recommendation 100 pg gDNA per sample Kit configuration 48 reactions Multiplexing capacity 16 libraries per run Number of primers >500 Number of amplicons 245 Average amplicon size 131 bp Amplicon overlap ≥3 bp Total library prep time 7 hours and 45 minutes Hands-on time 1 hour and 45 minutes
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.