Description
The ForenSeq mtDNA Control Region Kit offers convenient, efficient library prep for interrogating the control region of the mitochondrial genome (mtGenome) with minimal DNA input and optimum sensitivity. Leveraging proven ForenSeq chemistry, the kit produces a concise data set from the entire control region for direct upload to CODIS. Multiplexing capability boosts operational efficiency without increases to costs or time.
The ForenSeq mtDNA Control Region Kit links a powerful assay with industry-leading sequencing and integrates seamlessly with intuitive software for optimum laboratory efficiency. The ForenSeq mtDNA Control Region workflow is completely flexible and scalable for all throughput requirements, allowing laboratories to quickly generate complete mtDNA control region results for up to 48 samples simultaneously in less than two working days. For low throughput requirements, the kit’s 48 reactions maintain a 12-month shelf-life and for higher demand laboratories, a similar library preparation routine makes automation easy. Short mean amplicon size for maximum variant detection Optimized for inhibitor tolerance Flexible and scalable throughput Integrated workflow for optimum laboratory efficiency The high density of mutations concentrated in the mtDNA control region and the degraded nature of most forensic samples in which mtDNA analysis is used make optimal primer design challenging. The ForenSeq mtDNA Control Region kit is optimized to generate complete and actionable coverage, even for very low input amounts (see figure “The ForenSeq mtDNA Control Region system delivers extensive coverage across a wide range of DNA input amounts”). To improve performance on highly degraded material, the ForenSeq mtDNA Control Region kit uses more than 120 primers, designed from recently curated mtDNA variant and frequency data, generating 18 primary amplicons in the mtDNA control region. All primary amplicons are <150 bp in length, many < 100 bp – the lowest average amplicon size of any current commercial mtDNA assay system. All amplicons have a minimum overlap of 3 bp to avoid gaps in sequence following post run, bioinformatic trimming. A buffer system optimized for inhibitors that creates a robust amplification environment (see figure “An improved ForenSeq buffer system maximizes performance“). Feature Details Target size 1200 bp mtDNA control region DNA input recommendation 100 pg gDNA and mtDNA per sample Sample types DNA extracted from a range of degraded samples, including bone, teeth and hair shaft Recommended multiplexing capacity 3–48 libraries per run Number of primers >120 Number of amplicons 18 Average amplicon size 118 bp Amplicon overlap ≥ 3 bp Total library prep time 7 hours and 45 minutes Hands-on time 1 hour and 30 minutes
The PCR-based assay deploys small amplicons sourced from the latest mitochondrial (mtDNA) databases for enhanced variant detection. A tiled 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, while 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 48 samples in one run.
