Terminal restriction fragment length polymorphism profiling of bacterial flora derived from single human hair shafts can discriminate individuals
Introduction
Among the evidence that an offender leaves at a crime scene, human hairs are often found in criminal investigation [1], [2], [3]. Their judgment is mainly carried out by morphological observation using microscopy [1], [4] and human short tandem repeat (STR) typing [3], [5]. It is estimated that 50–150 strands of human hair are shed naturally every day [2], [6]. Therefore, there is some possibility of collecting an offender’s hair as forensic evidence, without their knowledge. If personal discrimination of the shed hairs were possible, it would be very helpful for identifying an offender, or for establishing an offender's action [1].
The human scalp contains 100,000–150,000 hairs on average [2], [7]. Of these, 90–95% are in anagen stage and the others (5–10%) are in telogen stage [8]. The telogen hairs have stopped growing, and apoptosis and keratinization of the hair root are progressing [2], [3], [6], [9]. As a result, it is easy for telogen hairs to fall out without external force due to their weak adhesion to the scalp [2], [6]. In general, the hair root is used for nuclear DNA extraction for human DNA typing [1], [2], [3], [5]. However, it is considered to be difficult to extract nuclear DNA from hairs recovered at a crime scene, because 90–95% of those hairs are at telogen stage [2], [7]. In fact, Bourguignon et al. found that enough DNA could be extracted from only 3.3% of 3242 telogen stage hairs from 27 volunteers for STR typing [3].
Because of these limitations, the mitochondrial DNA test is sometimes performed, since it is a more sensitive appraisal technique than STR typing [7], [10]. However, mitochondrial DNA testing has not been adopted in routine forensic examination in Japan because 1) there is no database like that for STR typing for comparison, 2) mitochondrial DNA shows maternal heredity, and 3) there is a possibility of the occurrence of an irregular heteroplasmy [6], [9]. Therefore, we focused on DNA derived from bacteria attached to the hair instead of human DNA from the hairs themselves.
It has been already reported that bacteria attached to human hair forms a complex flora [11], [12], [13]. For example, Tridico et al. performed metagenomic evaluations of bacteria on human hairs using next-generation sequencing data, and revealed that Corynebacteriaceae and Tissierellacea families were major bacterial taxa on human hairs [13]. In Japan, the terminal restriction fragment length polymorphism (T-RFLP) technique is currently the most applicable among all the general forensic laboratories [14]. We have already reported the possibility of discriminating an individual from the bacterial flora existing in their handprints using T-RFLP [15]. To date, there are no reports on discriminating an individual based on the bacterial flora on human hairs using T-RFLP. In preliminary experiments, we found that sufficient bacterial DNA could be recovered from a single shed hair. In this study, we aimed to analyze bacterial flora on single strands of human hair by T-RFLP to discriminate individuals as an alternative approach to STR typing or mitochondrial DNA judgment, without impairing the conventional inspection technique.
Section snippets
Samples and collection
Hair samples were collected from 24 Japanese adults (18 males and 6 females) who consented to take part in this study. None of the volunteers was taking medication during the experimental period. Samples of naturally shed or plucked hairs were collected while wearing nitrile gloves.
To examine the variation of T-RFLP patterns within an individual over time, a follow-up sampling from 16 volunteers was conducted after 6 months. The hair sample collections were carried out during 2 periods, April
Bacterial DNA yields from hair for T-RFLP analysis
We first investigated the yield of bacterial DNA extracted from each piece of hair. Hair roots and shafts from 50 hairs derived from 18 volunteers were analyzed separately. The average length of hairs used was 6.98 ± 4.99 cm, and the amount of bacterial DNA obtained from a hair root and a hair shaft were 40.06 ± 89.93 pg and 17.99 ± 18.30 pg, with the minimum amount of 0 pg and 1.32 pg, respectively. The average amount of bacterial DNA obtained from hair roots was twofold higher than that from hair
Discussion
Our previous study on T-RFLP bacterial profiling of handprints suggested that bacterial flora analysis is effective for discrimination among individuals [15]. Further to this, we aimed to establish a novel method of hair bacterial profiling, which could be routinely applied in crime scene analysis without any additional instrumentation. In this study, we investigated the possibility of individual discrimination using T-RFLP analysis of the hair bacterial floras by targeting 16S rRNA genes.
References (40)
Forensic hair morphology comparison -a dying art or junk science?
Sci. Justice
(2004)- et al.
A fluorescent microscopy- screening test for efficient STR-typing of telogen hair roots
Forensic Sci. Int. Genet.
(2008) - et al.
A quantitative assessment of a reliable screening technique for the STR analysis of telogen hair roots
Forensic Sci. Int. Genet.
(2013) - et al.
Short tandem repeat (STR) genotyping of keratinised hair part 1. Review of current status and knowledge gaps
Forensic Sci. Int.
(2005) - et al.
Short tandem repeat (STR) genotyping of keratinized hair part 2. An optimized genomic DNA extraction procedure reveals donor dependence of STR profiles
Forensic Sci. Int.
(2005) Microbial ecology of human skin in health and disease
J. Investig. Dermatol. Symp. Proc.
(2001)- et al.
T-Align, a web-based tool for comparison of multiple terminal restriction fragment length polymorphism profiles
FEMS Microbiol. Ecol.
(2005) DNA reviews: hair
Forensic Sci. Med. Pathol.
(2007)The quantitative classification of hair form and its application to the forensic comparison of Japanese head hair
Jpn. J. Technol. Ident.
(2003)- et al.
Sequencing of mtDNA in shed hairs: A retrospective analysis of material from forensic cases and a pre-screening method
Open Forensic Sci. J.
(2012)