"It is unfortunate that we live in a world where the best science is not always being pursued. Budgetary realities and concerns, regardless of whether they are manpower based or reagent based, force organizations to make compromises that they often do not prefer. Otherwise, the community would use an assay like the ForenSeq kits."
When DNA naturally degrades, whether due to exposure to the elements or just the age of the sample, it is typically split into fragments between 160 and 180 base pairs. When analyzing and genotyping highly degraded DNA evidence from a crime scene, traditional methods of short tandem repeat, or STR, analysis such as capillary electrophoresis are prone to fail due to amplicon size.
Bobby LaRue, an associate professor of forensic science at Sam Houston State University, has built his career looking at markers and marker systems that can return a strong power of discrimination, or low random match probabilities, with PCR amplicons smaller than 160 base pairs.
In this exclusive interview, we speak with LaRue about his hopes to expand and improve on the current go-to technologies for DNA evidence analysis and how a corporate partnership with Verogen is providing the university with access to comprehensive forensic analysis solutions.
Before entering into a career in forensic science, LaRue served in the US Army infantry. Here, he wondered how you could identify clandestine IED makers based on degraded DNA found on scattered bomb fragments. This inspiration has evolved into a desire to improve mixture deconvolution in complex DNA mixtures through advancements in current forensic methods and technologies, such as retrotransposable element markers.
With current dye-based systems, forensic labs are limited by the real estate available per dye channel. However, a massively parallel sequencing (MPS) approach doesn’t limit an analyst to just one workflow. For instance, in a sexual assault case, you may perform a Y-STR analysis because you only want to look at the male contributor. However, if you are not limited by the number of dye channels, you can look at the standard autosomal markers, the male-specific markers, ancestral informative markers, or phenotypic markers, along with any number of other aspects, all in one assay.
This is a particularly important consideration if you are working with a limited amount of sample. Ideally, a crime lab would not be limited to running only one type of analysis, as you would want to gather as much information as possible to use in downstream identification. Often labs must choose which marker they can include, and which markers they must do without, as each analysis consumes a portion of the limited sample. MPS allows labs to both amplify and analyze all the desired information simultaneously, enabling the use of smaller amplicons to cover the STRs - a difficult accomplishment for traditional capillary electrophoresis.
LaRue highlights that the main benefit of the ForenSeq kits is that they are not limited by the number of dye channels, explaining how "with the ForenSeq kits from Verogen you are only limited by the throughput of the instrument.”
The ForenSeq DNA Signature Prep Kit types all the standard CODIS core loci, the majority of the European markers, a number of biogeographical, identity-informative, and phenotypic markers, as well as single nucleotide polymorphisms (or SNPs).
Individually, SNPs don't have the discriminatory power that a highly polymorphic STR does, but, since you're not limited to the real estate on a dye channel with the ForenSeg kits, you can include several SNPs for additional resolution without any additional cost. LaRue explains: “We've typed rootless hair shafts, which in the past would not be considered for nuclear DNA testing, because of the high failure rate associated with STR typing. Despite this, we were able to get high enough SNP profiles with random match probabilities of 1 x 10-14 or 10-15, which is a pretty high-power discrimination.”
With this discriminatory power, analysts can readily distinguish between two individuals. In addition, when trying to generate an investigative lead, there are special biogeographical markers where, just from a DNA sample, you can tell whether or not this was left by somebody of Caucasian descent, East Asian descent, or even African descent by looking at the associated genetic profile.
In addition to its high-power discrimination, the ForenSeq kits show promise for the analysis of DNA mixtures. Capillary electrophoresis-based genotyping only looks at the size of a DNA fragment, not the individual sequence associated with it. So, if two individuals have different alleles by sequence, but with the same fragment size, they will stack and be indistinguishable from each other. However, when looking at something from a sequencing standpoint, you're able to see the individual sequences. From this, you can see if these size fragments have an internal SNP where you can distinguish between the two. LaRue goes on to say that: “if you conclude that 80% of the reads are from person A, and then 20% of the reads are from person B, you can conclude the same ratio with any PCR slippage artefacts, such as stutter products.”
The most exciting aspect of doing STRs on massively parallel sequencing platforms is the depth of coverage that they give to complex mixture interpretations.
Alongside the ForenSeq DNA Signature Prep Kit, LaRue’s lab has also beta-tested the ForenSeq mtDNA Control Region Kit and found this offered a vast improvement over traditional methods.
Traditional mitochondrial analysis uses Sanger sequencing, which is not easily done in a standard crime lab. The most commonly analyzed part of the mitochondrial genome is the control region, containing hypervariable regions commonly used for differentiating individuals. However, within these regions, there are long poly-C stretches that, as LaRue points out, “tend to wreak havoc on Sanger sequencing” and cause problems with read accuracy.
The depth of coverage that an MPS approach provides means that if you have one poor read, you have plenty of other reads that make up for this. LaRue explains: “The depth of coverage across the entire control region is not consistent, but there will be sequencing bias anytime you do anything with MPS. Verogen has managed to get the bias down to the point where you can effectively sequence with as little as one picogram of genomic DNA.”
Common objections to the use of MPS platforms are the cost and the turn-around time. Traditional capillary electrophoresis-based analyses can return results within 24 hours and are comparatively inexpensive for a single panel of markers. However, if the results come back inconclusive, either additional markers are needed or runs must be repeated, meaning the cost to the lab in both analyst time and money can increase significantly. For cases that would benefit from multiple marker types, such as autosomal and Y STRs, or samples where the DNA input is limited so repeated testing is difficult or impossible, then the ForenSeq kits offer a very time, sample, and cost-efficient alternative. In LaRue’s words: “With the ForenSeq kits you get everything and more all in one go.”
The key to success for the program at Sam Houston State University:
LaRue explains how collaborations with corporate partners provide them with a real advantage:
“We’re really fortunate, at Sam Houston, to receive a tremendous amount of support from our administration for corporate academic partnerships. The work that we do with multiple companies, including Verogen, grants us access to reagents and equipment that we wouldn't have the funding for otherwise.”
These partnerships also allow Sam Houston to assess these assays and kits to provide an unbiased opinion and third-party confirmation to the community that these kits work and that they provide a benefit to the field of research.
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