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Forensic DNA Analysis: Massively Parallel Sequencing Workflows

Massively parallel sequencing (MPS) offers a solution to the biggest challenges facing CE methods, such as distinguishing true allele variation from PCR artifacts, interpreting mixtures, and obtaining usable profiles from degraded DNA. However, adopting a new technology can have unique challenges. The PowerSeq® product family can help make the transition a smooth one.

PowerSeq® products enable target amplification of the mitochondrial control region and whole mitochondrial sequences. In addition, PowerSeq® products can amplify short tandem repeats (STRs) in both Y-chromosome and autosomal DNA to prepare Illumina® sequencing libraries for MPS analysis.

PowerSeq® Whole Mito System

Streamlined workflow for complete mtDNA analysis

The PowerSeq® Whole Mito System generates 161 small amplicons covering the entire region of the mitochondrial genome. The targeted regions for amplification are designed for an average size of 167bp to ensure optimal results from degraded samples. The system contains reagents for library preparation and a streamlined workflow that greatly reduces the number of steps and time required to produce libraries ready for sequencing.

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powerseq crm mtdna workflow mps

PowerSeq® CRM Nested System, Custom

Streamlined library prep for mtDNA analysis

The PowerSeq® CRM Nested System, Custom, generates 10 small amplicons covering the control region of the mitochondrial genome in one multiplex reaction. This technique improves the results for degraded samples. The workflow greatly simplifies library preparation by saving time, decreasing sample loss and reducing data variability.

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powerseq crm mtdna workflow mps

PowerSeq® 46GY System

Optimized panel of loci for STR analysis

The PowerSeq® 46GY System offers the same sensitivity as STR amplification kits. It can be used to prepare MPS libraries and generate sequencing data compatible with Illumina® sequencing technology. The kit enables identification of sequence variants in familiar autosomal and Y-STR loci, so that data generated are usable and interpretable with current guidelines.

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powerseq 46gy str analysis mps

PowerSeq® Quant MS System

Consistent library prep for STR analysis

The PowerSeq® Quant MS System is a qPCR-based system designed for quantitation of libraries prior to sequencing on the Illumina® MiSeq® system. Accurate library quantitation provides balanced representation of pooled libraries and reduces sequencing bias.

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powerseq quant ms library prep

Massively Parallel Sequencing for Forensic DNA Analysis

Traditionally, capillary electrophoresis (CE) has been the method of choice for analyzing short tandem repeats (STRs) for human identification. Although CE methods continue to evolve in terms of increasing sensitivity and the ability to work with degraded or poor-quality DNA, the technique still faces some challenges.

Next-generation sequencing (NGS), or massively parallel sequencing (MPS), enables the simultaneous analysis of hundreds of genetic markers, considerably more than current CE technologies. In addition to providing information on the size of the repeated regions, as CE does, MPS determines the underlying DNA sequence of each region. In doing so, MPS offers a solution to the biggest challenges facing CE methods, such as distinguishing true allele variation from PCR artifacts, interpreting mixtures, and obtaining usable profiles from degraded DNA.

A popular application of MPS in forensics is the sequencing of mitochondrial DNA—either the variable control region or the whole mitochondrial genome—with increased mixture deconvolution and heteroplasmy resolution compared to traditional methods. Although adoption of MPS workflows by forensic laboratories has been slow, the technology holds great promise for increasing the accuracy and throughput of forensic DNA analysis. Future advancements in MPS to further lower the cost will encourage more widespread adoption and consequent validation of forensic MPS methods.