Forensic Science International

Volume 329, December 2021, 111060

Assessment of the link between evidence and crime

scene through soil bacterial and fungal microbiome:
A mock case in forensic study
Sukriye Karadayı

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https://doi.org/10.1016/j.forsciint.2021.111060
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Abstract

In forensic studies, soil traces can be used to find clues to the origin of an unknown sample or the
relationship between a crime scene and a suspect and can provide invaluable evidence as they frequently
adhere to objects, with high persistence. In this study, it was aimed to investigate the potential of the
bacterial and fungal microbiome diversity of the soil to be used as legitimate evidence in the resolution of
homicide cases. Within the scope of a mock homicide case scenario, a total of 12 soil samples were
collected, including two evidence samples, three crime scene samples and seven non-crime scene related
control samples. Both bacterial and fungal microbiome profiles of these samples were analysed using
Illumina NovaSeq platform. The resulting sequences were analysed using QIIME 2 microbiome
bioinformatics platform. Beta diversity analysis was performed to determine the difference between
samples. In bacterial community analyses, it has been observed that it is difficult to distinguish evidence
samples and crime scene samples from control samples at phylum and class level, whereas differentiation
could be made at genus and species level. Fungal community analyses allowed to distinguish evidence
samples and crime scene samples from control samples at both phylum and class and genus and species
level. Principal coordinate analysis (PCoA) results showed that distance between evidence samples and
crime scene reference samples was closer to each other than non-crime scene related control samples. The
results of this study showed that bacterial and especially fungal DNA in soil has the potential to contribute
effectively to the resolution of forensic cases. Thus, it has been understood that it is possible to establish a
relationship between the case and the crime scene with the help of microbiome analyses on soil samples
obtained in homicide cases.

Introduction
To determine whether the suspect or suspects are murderers is difficult to resolve in the forensic
investigation process in homicide cases. In many forensic cases, biological materials such as blood, semen
and saliva are collected primarily from the victim and the offender during the evidence collection stage.
However, it is not always possible to obtain suitable amount of good quality DNA because of the failure to
find any human biological evidence, a certain period of time has passed since the crime, or the degradation
of samples collected. For this reason, there is always a need for new scientific techniques and methods in
order to establish the link between the offender and the crime or crime scene. In forensic science, soil can
provide invaluable confirmatory evidence in the resolution of forensic case because of its prevalence and
easy transferability based on Locard’s principle of exchange [1]. Soil, which can be characterised by wide
range of techniques, persistently adheres to an item of clothing, shoes, rubber, thus can be used to find clues
to the origin of an unknown specimen or to link between a crime scene and a suspect [2], contributing to
criminal justice processes as very strong evidence [3], [4], [5], [6], [7]. In addition, the unique nature of the
soil, including its mineral properties and vegetation, provides ideal form of trace evidence in forensic
investigations and is often overlooked by the offenders during their effort to conceal the evidence [8].

Soil evidence is one of the powerful tools frequently used to resolve criminal cases, especially in developed
countries [4]. Soil’s capacity of transfer to items such as shoes and clothing and its persistence may vary
depending on soil properties, mineralogy, and contact type [6], [9]. In addition, it is possible to encounter
soil layers or mixtures of different properties on these items because of its use before and after the crime. In
fact, soil constitutes the world’s largest phylogenetic and functional diversity per volume with values
between 104 and 107 per gram [10], [11]. Components or contaminants in the soil such as glass, pollen, fibre
or plant residues and the colour of the soil provide valuable evidence for the resolution of forensic cases
[13], [14], [12]. If we consider the complexity of the chemical and biological composition of the soil, the
level of knowledge obtained enables a fingerprint specific to the study area [3]. Standard forensic soil
analysis is focused on examining the physical appearance and chemical composition of the soil, including
pH, colour, texture, particle size, mineralogical and geophysical studies [5], [6], [7]. However, these methods
have some disadvantages such as being quite costly, requiring a great deal of time to interpret the results
and need for experienced soil experts. In addition, many of the methods used to analyse soil rely on
imprecise visual comparisons and are unfortunately controversial in the forensic community, as some
studies often fail to achieve statistical significance in the relationships between questioned and known
samples. Random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism
(AFLP), terminal restriction fragment length polymorphism (TRFLP), amplified ribosomal DNA restriction
analysis (ARDRA), ribosomal intergenic spacer analysis (RISA), length heterogeneity-polymerase chain
reaction (LH-PCR), and denaturing gradient gel electrophoresis (DGGE) which are investigated to be used in
forensic soil analysis in recent years has both advantages and disadvantages [15]. Indeed, it is clear that
more definitive, higher resolution data should be produced for the use of soil analyses in forensic science
[16]. On the other hand, by connecting criminals to a crime scene, microbiome research provides a wide
variety of evidence and can ultimately develop tremendous forensic potential for case resolutions [17]. In
this context, since the microbial structure and composition of the soil can differ significantly between
habitats, many researchers have recommended next generation sequencing (NGS) as an objective method to
individualise soil samples [16], [18], [8]. Soil DNA sequencing studies have revealed that microbiome
community structures are greatly influenced by soil type [18], [19], suggesting that soil microbial profiling
can be improved for forensics [20].

Since the community structure of bacteria may vary depending on the characteristics of the soil, seasonal
changes, vegetation and environmental conditions in the region, bacteria have a highly region-specific DNA
profile [21], [22]. Soil DNA fingerprinting analysis for forensic studies previously included microbiome
studies targeting only the bacterial 16S ribosomal RNA (rRNA) gene region [23], [16]. However, in
subsequent studies, it has been shown that the discriminatory power of the fungal microbiome profile of
the soil is also high [24], [25]. In addition, high stability of fungal profile against changing soil conditions
such as drying has been demonstrated [26].

Forensic soil analyses are usually done by comparing interrogated samples with samples of known origin to
assess whether there is similarity (inclusion) or difference (exclusion) between the evidence at the crime
scene and the suspect/suspect samples [20]. In a forensic case, it is crucial to primarily identify the evidence
at the crime scene, followed by accurate sampling, store, identification, individualisation, interpretation and
then establish its linkage with the crime for reconstructing the crime correctly. When this process is carried
out in reverse, the importance of reconstruction of crime scene scenarios, of which all the conditions that
may affect the crime are known, cannot be denied to test the power and reliability of the evidence and
analysis.

In this study, it was aimed to investigate the potential of the bacterial and fungal microbiome diversity of
the soil to be used as legitimate evidence in the resolution of homicide cases. In this context, with the help
of a forensic murder case scenario, the determination of microbiome diversity by sequencing the bacterial
(16S rRNA) and fungal regions (internal transcribed spacer 1 [ITS1]) of soil evidence, and analytical
similarities between bioinformatic analyses and evidence were revealed.

Section snippets

Mock homicide scenario

Within the scope of the study, a homicide case was designed to investigate the potential of the microbiome
structures of soil samples as evidence. According to this scenario, “In the Gürpınar village of Beylikdüzü region
in Istanbul, a shepherd reported that a man’s body was found in a sack in a dried-up stream bed in a desolate
area. In the examination carried out on the corpse and clothes on 19 June 2020 at the crime scene, heavily dried
soil samples were found on the left knee of the…

Results

In this study, 12 soil samples were collected and analysed, including two evidence samples (E1, E2), three
crime scene samples (CS1-CS3) and seven control samples outside the crime scene (C1-C7).…

Discussion

Soil can provide clues to help trace the origin of evidence gathered in forensic studies, because of its
heterogeneity and transferability and as it contains both bacterial and fungal structures. It contributes to the
comparison of samples collected from an object with samples taken from the crime scene. The most
important feature of bacterial profiling studies in forensic soil analysis is its effectiveness in differentiating
different habitats [16]. Bacterial DNA analyses are beneficial to…

Conclusions

The presented study obviously confirms the potential usage of bacterial and fungal DNA as a valuable tool
for forensic sciences to discriminate soil samples. Thus, it is possible to establish a relationship between the
case and the crime scene with the microbiome analyzes performed on soil samples that are likely to be
obtained in murder cases. It would be more beneficial to evaluate bacterial and fungal microbiome
structures together in establishing the connection between evidence samples and…

Funding

This research project was supported by Altınbaş University Scientific Research Fund. Project Number:
PB2018-GÜZ-SHMYO-2.…

CRediT authorship contribution statement

CSukriye Karadayı: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources,

Data curation, Writing – original draft, Writing – review & editing, Supervision, Project administration,
Funding acquisition.…

Declaration of Competing Interest

The author declares that they have no known conflicting financial interests or personal relationships that
could have appeared to influence the work reported in this paper.…

Acknowledgment
The author acknowledges Dr. Beytullah Karadayı for his forensic scientific contribution and Dr. Tülin
Arasoğlu and Dr. İlkgül Akmayan for their molecular genetic scientific contribution.…

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