A guest blog by Ryan Gutierrez, PhD candidate, Sam Houston State University
Though commonly found at crime scenes, hair’s reputation as a low-quality forensic sample results in this tissue being underutilized as evidence. Rootless hair shafts, with their low template and degraded DNA, are particularly challenging. Alternative forensic testing workflows, however, can recover genetic material from even rootless hair—achieving probative DNA results in cases where hair evidence plays a major role.
In my lab at Sam Houston State University, we used a novel hair extraction chemistry in conjunction with a variety of methods for DNA analysis. We found that alternative extraction and analysis workflows can significantly improve allele recovery from hair samples.
The Root of the Problem
Rootless hair shafts pose two major concerns in forensic settings. The process of hair growth itself results in the first concern—the keratinization process that creates the hair shaft often lyses the nucleus. This results in the degradation of nuclear DNA in hair samples across the board. Secondly, hair samples can be limited in size, often consisting of only a small length of hair, with few if any intact cells containing DNA.
This combination of low template and high degradation combine to make rootless hair shafts a highly challenging sample source. When nuclear DNA can be recovered, it’s highly variable whether this genetic material can be successfully amplified with traditional STR analysis.
In contrast to nuclear DNA, mitochondrial DNA is more readily recoverable from hair shafts, where mitochondrial DNA has greater copy numbers. Recoverability also results from the additional mitochondrial membrane, which somewhat protects this genetic material from degradation.
This recoverability makes mitochondrial sequencing more reliable than STR amplification when it comes to challenging samples. There are, however, some disadvantages. Mitochondrial inheritance patterns result in a low power of discrimination when comparing random individuals—and no power to discriminate between maternally related individuals.
An Alternative Approach
There is, however, another option. Highly degraded, low-template nuclear DNA samples may be unsuitable for STR analysis, but may yield good results with alternative methods. Our research team—including Bobby LaRue, Ph.D., Associate Professor of Forensic Science, and myself—decided to evaluate a novel tool for extracting nuclear DNA from rootless hair shafts.
Recently developed, the InnoGenomics InnoXtract kit is formulated to increase recovery of small fragments of DNA from challenging samples. Following lysis, this bead-based extraction method produces DNA extracts that are free from inhibitors and capable of being used for multiple downstream applications. Preliminary studies have used extracts from the InnoXtract kit to reliably perform PCR amplification for use with both capillary electrophoresis (CE) and massively parallel sequencing.
Rootless hair samples extracted with the InnoXtract chemistry have been used by researchers with the CE-based GlobalFiler, InnoTyper 21, and Big Dye Direct Sequencing Chemistry. Sample extracts were also amplified with the ForenSeq DNA Signature Prep Chemistry and Mitochondrial Control Region Solution for use with the MiSeq FGx.
Allele recovery from our data set averaged 50% when using InnoXtract and InnoTyper 21 on untreated rootless hair shafts, compared to 22% allele recovery with traditional STR chemistry.
Probative Results from Challenging Samples
Success with rootless hair shafts depends on the recovery of highly degraded DNA. Strategies that incorporate PCR to amplify small DNA fragments and are developed for use with degraded templates will lead to more successful outcomes, compared to traditional STR amplification. While sacrificing the polymorphic nature of STRs, small amplicon bi-allelic-based assays such as InnoTyper 21 can recover genetic information that’s orders of magnitude more discriminatory than mitochondrial sequencing.
Traditional extraction chemistries are optimized to recover DNA for use with STR-based methods. While these assays are effective for many samples, recovery of DNA fragments too degraded for STR analysis is important when attempting alternative testing workflows on degraded and low-template DNA.
When used as an additional option in cases where mitochondrial sequencing is insufficient, samples amplified with an additional nuclear-based chemistry can provide the discriminatory power lacking in mitochondrial data.