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The Power of PowerSeq® MPS Products

Ann MacPhetridge, Promega

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The Benefits and Limitations of Massively Parallel Sequencing

While capillary electrophoresis technology (CE) for analyzing short tandem repeats (STRs) is still the workhorse in forensic casework, we all recognize that STR technology poses certain limitations in samples of low quantity or compromised quality, such as mixtures or degraded samples. These limitations can be overcome by massively parallel sequencing (MPS), where multiple targets can be queried in a single assay, because shorter amplicons provide better interpretable data. In addition, mixture deconvolution is easier due to greater depth of allele and sequence information.

What is the basic difference? CE analysis detects variants based on size differences whereas MPS detects both allele size and sequence variation, adding another level of discrimination. Further, single-nucleotide polymorphisms (SNPs) flanking the repeat sequence can identify additional alleles, contributing to discrimination power. For example, sequencing of Y chromosome loci can help distinguish between mixed male samples from the same paternal lineage and, therefore, provide valuable information in decoding mixtures that contain more than one male contributor. Also, since MPS technology is not limited by real estate, all primers in an MPS system can target small loci, maximizing the probability of obtaining a usable profile from degraded DNA.

Both CE and MPS workflows start with sample collection, purification and quantification of DNA. In the CE workflow, we amplify STR loci using fluorescent primers on a thermocycler. The resultant amplicons are separated on a CE instrument, and identified by size and label to generate a DNA profile. In MPS, the targets are amplified with unlabeled primers and go through a series of enzymatic steps to prepare a library. Libraries pooled from multiple samples are normalized and loaded on a flow cell, placed on a sequencer (e.g., MiSeq® instrument) and analyzed.

MPS systems enable simultaneous analysis of forensically relevant genetic markers to improve efficiency, capacity, and resolution—with the ability to generate results on almost 10-fold greater genetic loci than the current technology. Many MPS kits provide additional primer sets for identification of ancestry, geographical and phenotypic SNPs, but there are distinct hurdles to adopting them in regular casework. There is simply not enough population data in the database to provide a true assessment of discrimination power and, more importantly, the community is yet to arrive at a consensus for reporting guidelines. Moreover, many countries do not allow use of phenotypic and ancestry SNPs in casework.

An Introduction to the PowerSeq® 46GY System

Since the MPS technology itself is complex, simpler kit configurations are easier for labs to adopt for their casework without the burden of analyzing supplementary, unestablished markers. The PowerSeq® 46GY System combines unlabeled primers for PowerPlex® Fusion and PowerPlex® Y23 in a single kit and therefore can be used to generate sequence data for existing known STR loci. These loci are widely accepted in the forensic community where adequate population data and reporting guidelines are in place—making the transition to MPS a bit easier.

The PowerSeq® 46GY System has the largest combination of autosomal and Y-STR loci in a single kit to generate sequence data and improve your MPS workflow. Amplicons for each locus are designed to be in the range of 140–300bp. The loci included are CODIS core and European Standard Set (ESS) loci, Amelogenin, and the Y-STR loci included in the PowerPlex® Y23 System.

Autosomal: CSF1PO, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D1S1656, D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, Penta D, Penta E, TH01, TPOX, vWA

Y-STR: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS481, DYS533, DYS549, DYS570, DYS576, DYS635, DYS643, Y-GATA-H4

Other: Amelogenin

What samples would truly benefit from MPS? Mixture samples, undoubtedly. The benefit of MPS is also exemplified in cases where the samples are highly degraded or the only samples available are teeth, bones and hairs without a follicle. By adding a sequencing component to the allele length component of CE technology, MPS resolves the current greatest challenges in forensic DNA analysis—namely identifying allele sharing between contributors and PCR artifacts, such as stutter. Here we have a female sample that shows allele 28 and 30 for D21S11 locus. When this female sample is mixed with a male sample in the ratio of 20:1, we can see additional data, but unlike CE, we can also easily identify that alleles 29 and 31.2 belong to the male contributor, despite the fact that allele 29 is also a stutter location for allele 30. The individual contributions to allele 29 can be identified by sequence—something that would not be possible when analyzing CE data.

By offering the same sensitivity, robustness, inhibitor tolerance and locus balance as PowerPlex® STR Systems, the PowerSeq® 46GY System enables identification of sequence variants in familiar autosomal and Y-STR loci, enabling laboratories to adopt MPS technology for forensic casework more easily.

To learn more about the PowerSeq® 46GY System as well as our other PowerSeq® products, visit

To request a kit to evaluate in your laboratory, please contact your local Promega representative.