Australian Forensic Wildlife Laboaratory

Abstract
Background: Mammalian hairs are one of the most ubiquitous types of trace evidence collected in the course of
forensic investigations. However, hairs that are naturally shed or that lack roots are problematic substrates for DNA
profiling; these hair types often contain insufficient nuclear DNA to yield short tandem repeat (STR) profiles. Whilst
there have been a number of initial investigations evaluating the value of metagenomics analyses for forensic
applications (e.g. examination of computer keyboards), there have been no metagenomic evaluations of human
hairs—a substrate commonly encountered during forensic practice. This present study attempts to address this
forensic capability gap, by conducting a qualitative assessment into the applicability of metagenomic analyses of
human scalp and pubic hair.
Results: Forty-two DNA extracts obtained from human scalp and pubic hairs generated a total of 79,766 reads,
yielding 39,814 reads post control and abundance filtering. The results revealed the presence of unique
combinations of microbial taxa that can enable discrimination between individuals and signature taxa indigenous
to female pubic hairs. Microbial data from a single co-habiting couple added an extra dimension to the study by
suggesting that metagenomic analyses might be of evidentiary value in sexual assault cases when other associative
evidence is not present.
Conclusions: Of all the data generated in this study, the next-generation sequencing (NGS) data generated from
pubic hair held the most potential for forensic applications. Metagenomic analyses of human hairs may provide
independent data to augment other forensic results and possibly provide association between victims of sexual
assault and offender when other associative evidence is absent. Based on results garnered in the present study, we
believe that with further development, bacterial profiling of hair will become a valuable addition to the forensic
toolkit.

An initial investigation of the application of laser scanning confocal microscopy to the examination of hairs and fibers has been conducted. This technique allows the production of virtual transverse and longitudinal cross-sectional images of a wide range of hairs and fibers. Special mounting techniques are not required; specimens that have been mounted for conventional microscopy require no further treatment. Unlike physical cross-sectioning, in which it is difficult to produce multiple cross-sections from a single hair or fiber and the process is destructive, confocal microscopy allows the examiner to image the cross-section at any point in the field of view along the hair or fiber and it is non-destructive. Confocal microscopy is a fluorescence-based technique. The images described in this article were collected using only the autofluorescence exhibited by the specimen (i.e. fluorescence staining was not necessary). Colorless fibers generally and hairs required excitation at 405 nm in order to stimulate useful autofluorescence; longer wavelength excitation was suitable for dyed fibers. Although confocal microscopy was found to be generally applicable to the generation virtual transverse cross-sections from a wide range of hairs and fibers, on some occasions the autofluorescence signal was attenuated by heavy pigmentation or the presence of an opaque medulla in hairs, and by heavy delustering or the presence of air-filled voids in the case of fibers. In these situations only partial cross-sections were obtained.

The large extinct megafaunal species of the Late Pleistocene, Mammuthus primigenius (woolly mammoth) and Coelodonta antiquitatis (woolly rhino) are renowned for their pelage. Despite this, very little research has been conducted on the form and function of hair from these iconic species. Using permafrost preserved hair samples from seven extinct megafaunal remains, this study presents an in-depth microscopical characterisation of preservation, taphonomy, microbial damage, pigmentation and morphological features of more than 420 hairs. The presence of unique structural features in hairs, from two extinct megafauna species, such as multiple medullae and unparallelled stiffness suggests evolution of traits that may have been critical for their survival in the harsh arctic environment. Lastly, despite popular depictions of red-haired and/or uniformly coloured mammoths, a closer examination of pigmentation reveals that mammoth coats may have exhibited a mottled/variegated appearance and that their ‘true’ colours were not the vivid red/orange colour often depicted in reconstructions. Insights gained from microscopical examination of hundreds of extinct megafauna hairs demonstrae the value of extracting as much morphological data as possible from ancient hairs prior to destructive sampling for molecular analyses.

Background: Mammalian hairs are one of the most ubiquitous types of trace evidence collected in the course of forensic investigations. However, hairs that are naturally shed or that lack roots are problematic substrates for DNA profiling; these hair types often contain insufficient nuclear DNA to yield short tandem repeat (STR) profiles. Whilst there have been a number of initial investigations evaluating the value of metagenomics analyses for forensic applications (e.g. examination of computer keyboards), there have been no metagenomic evaluations of human hairs—a substrate commonly encountered during forensic practice. This present study attempts to address this forensic capability gap, by conducting a qualitative assessment into the applicability of metagenomic analyses of human scalp and pubic hair. Results: Forty-two DNA extracts obtained from human scalp and pubic hairs generated a total of 79,766 reads, yielding 39,814 reads post control and abundance filtering. The results revealed the presence of unique combinations of microbial taxa that can enable discrimination between individuals and signature taxa indigenous to female pubic hairs. Microbial data from a single co-habiting couple added an extra dimension to the study by suggesting that metagenomic analyses might be of evidentiary value in sexual assault cases when other associative evidence is not present. Conclusions: Of all the data generated in this study, the next-generation sequencing (NGS) data generated from pubic hair held the most potential for forensic applications. Metagenomic analyses of human hairs may provide independent data to augment other forensic results and possibly provide association between victims of sexual assault and offender when other associative evidence is absent. Based on results garnered in the present study, we believe that with further development, bacterial profiling of hair will become a valuable addition to the forensic toolkit.