Forensic evidence and research about physical evidence in forensic, Study notes of Forensics

Download Forensic evidence and research about physical evidence in forensic and more Study notes Forensics in PDF only on Docsity! TOPIC : Establishment of identity of individual Sub: Forensic science Sem: 10 Div.: A Name: Fiza Bangali: 07 Mansi Dave: 14 Sangeeta Meena: 29 Nehal Sathvara: 45 1 INDEX NO. TOPIC Page no. 1 Foot print 03- 07 2 Blood Grouping 07 - 13 3 Skin and Hair 14 - 30 4 Physical Peculiarities 31 – 35  Introduction of footprint 2 case of Ram Singh and others v. State of Rajasthan and others, it has been ruled by the Court that evidentiary value of footprints are not well-established principle and in absence of other strong and reliable evidence that points towards the accused’s identification then footprint evidence cannot be used in order to conclusively prove any finding and the same position had been reiterated many times that the evidence of footprints are not a reliable source of evidence. Moreover, in the case of Sunder and others v. State of Rajasthan, the court held that it needs to be assured that the footprints collected are genuine and the whole procedure has been followed and no tampering has been done at any stage. Following it judges would also look up to other reliable evidence and circumstantial evidence and discern whether all such evidence points towards the guilt of the accused or a complete chain of evidence has been built up which gives some conclusive finding. Footprints evidence is used as circumstantial evidence and thus cannot alone determine the guilt of a person like fingerprints or DNA evidence. Footprints evidence are in most of the cases seen in the light of other evidences and their reliability are dependent on the direction in which it is pointing, meaning to say that if all the evidences are pointing towards a single person and footprint evidences also point toward the same person then such evidence would be accepted as reliable but if it proves contrary then it may be disregarded or same importance may not be attached to those evidences.  Critical Analysis of Footprint Evidences The aim of criminal law is to find out the actual criminal and prevent any erroneous convictions of innocent people. The Legal system generally relies on the evidence while supporting the case. Individualization forensic evidence aims to be scientific in nature both at the time of recovery and analysis. If forensic evidence is based upon reliable scientific methodology it paves the way for accuracy in the analysis of evidence or report finding. It is very important for any forensic evidence to rely on a scientific test which is objective in nature but footprint evidence lacks any scientific approach and thus it fails in meeting the demand of law. The footprint analysis involves a procedure where an object that is known like a shoe is compared with the partial or complete impression that was taken from the crime scene. Then the expert has to assess whether the impression found on the crime scene and known object has the same source or not. The whole procedure is done in a stepwise manner, first is the comparison of class characteristics and second is the identification of the characteristics. The whole procedure lacks the appropriate empirical studies for supporting the association of shoeprint with the known print done on the basis of identifying marks. It is very ambiguous to find out the accuracy of the examiner who identifies the features that are commonly present in both the impression and the shoe. It is difficult to find their range of error in distinguishing between the same features and assigning a probative value on such evidence is the toughest. 5 Footprint evidence is based upon the sense of probabilities which comes from experience. The experts may be in many cases true but it is very difficult to cancel out the biases which naturally come while making any experience based judgment and mostly in such cases where any feedback mechanism is absent to evaluate such erroneous judgement. The problem increases when experts have to deal with unique impression evidence. It is true that thousands of judgments made by an expert develops a sense of judgment and experience but the fact still remains the same that there is no scientific data on the basis of which degree of similarity to come on a result can be observed. The report given by the President's Council of Advisors on Science and Technology (PCAST) has made a conclusion that the fundamental issue is not of consistency but it is accuracy. PCAST also states that there is no empirical study that can conclusively prove or lay down foundational validity of any analysis for associating shoe prints on the basis of specific identifying marks. Conclusion of footprint evidence is neither supported by any meaningful evidence nor has its accuracy been estimated. The trail concludes that footprint evidence is not scientifically valid. Another major challenge that can be listed down is that only a limited number of databases exist which can be used while classifying the pattern of footwear outsole. There are insufficient studies on the manufacturing characteristics or about the frequency of the outsole pattern. For instance the shoe manufacturer neither provides the details about the number of shoes existing with a particular design nor do they tell about the shoes sold of one particular design. Considering the fact that there are a number of counterfeit shoes in the market which are a replication of the design of the outsoles which are belonging to popular brands. Another problem is that one is not aware of the time for which a person keeps that pair of shoes and all the factors in combinations makes it impossible to estimate the number of shoes belonging to a particular model existing among the population at a time.  Conclusion The footprint evidence has been overlooked and has been considered as weak evidence due to many reasons. Major ones that can be laid down are that there is a lack of education and training in properly collecting, searching, and preserving the evidence of footprints, and secondly, the evidence has been misunderstood and overlooked. The reason behind the failure of footprint evidence in the area of solving crime can be listed down, firstly that people don’t believe that impression can be found after people have walked over the crime scene, and secondly, it may be due to incomplete searches of the scene by the investigating agencies, thirdly because of weather conditions and fourthly when the impression has been destroyed intentionally. Examination of footprints relies mostly on the traditional comparison that is done by side by side ways method. 6 Eye tracker which is a scientific technology can be used to enhance footwear comparison. Eye tracker allows the examiner to track their own gaze while finding the similarities in question and known impression or evaluating the footprint evidence. A heat map is constructed through the process which allows the examiner to find out the areas of emphasis. Footprints as evidence can lead us in the journey of investigation. For it to be effective, it is very important that the most reliable method is used for the purposes of analysis and all the above mentioned issues and loopholes shall be resolved in order to give push to a scientific investigation and forensic science.  Blood grouping  The concept of Blood grouping was first discovered by Karl Landsteiner in 1901, who was an Austrian-American immunologist and pathologist. He received Nobel Prize in Physiology or Medicine in 1930 for this discovery.  His discovery helps us to determine blood groups and thus opened a way for blood transfusions that can be carried out safely.   There are present two types of Blood grouping as forward grouping and Reverse grouping.  The forward blood grouping is defined as using a known source of antibodies to detect the antigens on the red blood cells.   While Reverse grouping is defined as using the reagent cells with known ABO antigens and testing the patient’s serum for ABO group antibodies.  The ABO and Rh are the major, clinically significant, and the most important of all the blood group systems  The ABO blood groups are classified into four groups based on the presence or absence of two inherited antigenic substances on the surface of red blood cells (RBCs).  The four major ABO blood groups are  “A” group, “B” group, “AB” group, “O” group.  Blood group A contains antigen-A and antibody-B, Blood group B contains B-antigen and antibody-A, Blood group AB contains antigen-A and antigen-B and no antibodies, and Blood group O has no antigen and contains antibodies anti-A and anti-B.  The Rhesus system (Rh) is the second most important blood group system in humans. The most significant and immunogenic Rhesus antigen is the RhD antigen.   The Rh factor is present on the surface of RBC in most people. It is a type of antigen and those who have it are called Rh+. Those who lack this antigen are called Rh-.   The Rh antibodies are absent in the blood of those persons who have Rh-. But they can produce Rh antibodies if they get blood from a Rh+ person, whose Rh antigens can induce the formation of Rh antibodies (as the immune system is triggered by the presence of an unknown antigen in the system).  The  Rh+ person can receive blood from an Rh- person without any problem.  The Rh system consists of two allelic genes such as; RhD and RhCE 7 4. Mix them thoroughly on both slide. 5. Observe the agglutination on the slides. The Tube method 1. Take two tubes, one for the patient and one for the control. 2. Add one drop of Anti-Rh D in patient tube and one drop of albumin in control tube. 3. Add one drop of 2% – 5% patient’s RBC suspension on both slide. 4. Mix thoroughly and then centrifuge them. 5. Gently resuspend the sample. 6. Observe for the agglutination. Rh Blood Grouping Result Result for Slide Method  Positive Result: If the patient sample shows agglutination and the control shows suspension.  Negative Result: If both patient and control sample shows suspension. Result for Tube Method  Rh-D Positive: Agglutination in patient’s tube; and smooth suspension in control tube.  Rh-D Negative: Smooth suspension in both tubes. Interpretation  If agglutination is observed when blood is mixed with Anti A reagent, then the individual is said to have blood group “A”.  If agglutination is observed when blood is mixed with Anti B reagent, then the individual is said to have blood group “B”.  If agglutination is observed when blood is mixed with Anti A and Anti B reagent, then the individual is said to have blood group “AB”.  If no agglutination is observed when blood is mixed with Anti A and Anti B reagent, then the individual is said to have blood group “O”. 10  If agglutination is observed when blood is mixed with Anti RhD reagent, then the individual is said to have “+ve” Rh factor.  If no agglutination is observed when blood is mixed with Anti RhD reagent, then the individual is said to have “-ve” Rh factor. Application of blood grouping  The blood grouping is used during the blood transfusion.  It also used for paternity disputes.  Used to detect hemolytic disease of newborn.  Susceptibility to various diseases Ex: O group- peptic ulcer.  Used for part of Health check-up and job, driving licensing, etc. Precautions  Read the entire procedure before preceding with the experiment.  During the experiment wear gloves.  Make sure the slide is clean and dry prior to use.  Avoid touching the antisera reagent dropper to the blood sample.  The result of the reaction should be interpreted immediately after mixing.  To prevent a false result avoid the intermixing of the antisera reagents while performing the experiment.  Classical Strategies in BG Typing In routine clinical analysis, there is a wide range of established procedures and practices for blood typing, where nearly all of them deal with the formation of agglutinates. Even though some of these classical methods are not highly sensitive, nonetheless, they still hold importance in ABO grouping tests. There is a wide range of blood typing techniques, which differ from each other in terms of sensitivity, reagents and equipment required, the time of operation and throughput analysis. Herein, we describe some general approaches of blood grouping along with their inbuilt advantages and drawbacks. 2.1. Slide Method The slide test is relatively the least sensitive method among others for BG determination, but due to its prompt results, it is very much valuable in emergency cases. In this method, a glass slide or white porcelain support is divided into three parts, as for each part, a drop of donor or recipient blood is mixed with anti-A, anti-B and anti-D separately. The agglutination or blood clumping pattern can be visually observed from which the ABO and rhesus D (RhD) type of 11 blood can be determined. The test completes in 5–10 min and is inexpensive, which requires only a small volume of blood typing reagents. However, it is an insensitive method and only useful in preliminary BG matching for getting an early result. The test cannot be conducted for weakly or rarely reactive antigens from which the results are difficult to interpret, and additionally, a low titer of anti-A or anti-B could lead to false positive or false negative results. Although the slide test [is useful for outdoor blood typing, it is not reliable enough for completely safe transfusion. 2.2. Tube Test In comparison to the slide test, the tube test is more sensitive and reliable; therefore, it can be used conveniently for blood transfusion. In this method, both forward (cell), as well as reverse (serum) grouping is carried out. The forward grouping suggests the presence or absence of A and B antigens in RBCs, whereas reverse grouping indicates the presence or absences of anti-A and anti-B in serum. In forward grouping, blood cells are placed in two test tubes along with saline as a diluent media, and then one drop of each anti-A and anti-B is added separately in these samples. These tubes are subjected to centrifugation for few minutes, and then, the resultant matrix is gently shaken for observing agglutination. For precise blood grouping, the two tubes can be categorized according to the extent of blood clumping. The purpose of centrifugation is to ensure enhanced chemical interactions, particularly for weaker antibodies to react, thus leading to agglutination. Some potentiators could also be added to promote the agglutination; moreover, the long incubation of tubes also favors these reactions without drying of the test samples. In a similar fashion, reverse grouping can be performed, as here, the blood serum is treated against RBC reagent groups of A1 and B, and the subsequent agglutination pattern is monitored. The grading of agglutinates in both forward and reverse grouping is useful in comparing the difference in the strength of hemolysis reactions. In general, the tube method is much more sensitive than the slide test and requires a low volume of reagents, and some unexpected antigens can also be detected; therefore, it is a better option for safer transfusions. However, in infants, reverse grouping is somewhat difficult to perform, since they produce insufficient amounts of antibodies to be determined. 2.3. Microplate Technology Among classical methods, microplate technology is a further step towards more sensitive and fast blood typing analysis with the feasibility of automation. In this technique, both antibodies in blood plasma and antigens on RBCs can be determined. Typical microplates consist of a large number of small tubes that contain a few µL of reagents, which are treated against the blood samples. Following centrifugation and incubation, the subsequent agglutination can be examined by an automatic read out device. The microplate technique was first introduced in early 1950s; however, since then, considerable developments have been made in the design to improve the performance. The foremost advantage of microplate technology is its fast response, low reagent volumes and high throughput analysis. Apart from microplates, gel cards or strips can also be used for blood grouping in modern immunoassay machines. 12 tools, bottles, cups, victim’s skin, bite marks, lip prints, and drug paraphernalia (Kapoor and Chowdhry, 2018). Accepted, presumptive, and confirmatory tests of forensic samples based on saliva are available (Old et   al., 2009 ; Virkler and Lednev, 2009). However, some of these tests, particularly the presumptive tests, are often not specific to saliva. Other tests cannot differentiate vaginal fluid from saliva which may serve as a significant piece of evidence in reconstructing cases related to sexual assault. In addition, saliva may be detected in trace levels and the protein of interest for these tests may not be adequate enough. However, saliva contains microorganisms, which facilitates forensic investigations when combined with longstanding salivary biomarkers (Leake et   al., 2016 ). We included 19 samples of saliva obtained from five different countries listed in the FMD (Table   1 ), and 11 samples from each of the 11 countries, which are not included in the FMD (Table   2 ). Different geographical and environmental factors, such as diet, elements of hygiene, humidity, climate, temperature and oral disease, can affect the composition of microbial communities (Li et   al., 2014 ). Several studies of saliva have demonstrated a potential geographic signature involving the oral microbiome (Nasidze et   al., 2009a ; Clarke et   al., 2017 ). Saliva samples obtained from India, Italy, Japan, South Korea, and the USA were used in the FMD data. Streptococcus were the main bacteria in samples derived from most countries (Figure   2 ). Rothia was the highest in prevalence among Japanese; Prevotella was the highest in India and Italy; and Neisseria was the predominant salivary microbe among Italians and South Koreans. Takeshita et   al. (2014)  analyzed healthy South Koreans and Japanese and found that the salivary microbiome of Koreans harbored higher proportions of Neisseria, Haemophilus, and Porphyromonas and lower proportions of Prevotella and Veillonella compared with those in the Japanese population. Additionally, the geographical location had a remarkable effect on salivary microbiota, more than age, gender, or smoking, although the smoking status had a significant effect on the microbiome. In China, the salivary microbiome was dominated by 12 genera: Streptococcus, Neisseria, Haemophilus, Prevotella, Porphyromonas, Veillonella, Ge mella, Rothia, Granulicatella, Fusobacterium, Actinomyces, and Alloprevotella. Among them, Haemophilus and Prevotella were the most abundant genera in healthy individuals (Wu et   al., 2018 ). In Yokohama in Japan, the FMD data showed that Fusobacterium was the main genus (Table   1 ). However, when the Japanese state or city was excluded, the main genus was Rothia. Mashima et   al. (2019)  analyzed the salivary microbiome in rural Thai children 15 grouped into five clusters (east, west, south, north, and central) based on economic, food, and lifestyle factors and reported significant differences between Veillonella and Prevotella among the geographical regions (p < 0.05). In addition to their abundance, the presence of V. parvula, R. aeria, and R. dentocariosa indicated potential deterioration in oral hygiene, which also relates to dental caries history. Accordingly, individuals residing in Yokohama may exhibit comparatively better dental hygiene than the other populations. Also, Prevotella was the most common and abundant bacterium in East Asia (South Korea, Japan, China, and Thailand). Leake et   al. (2016)  reported the preponderance of eight major genera in the saliva samples of Swiss population: Streptococcus, Neisseria, Prevotella, Haemophilus, Veillonella, Porphyromonas,  Rothia, and Fusobacterium. However, despite the geographic proximity between Germany and Switzerland, no common genera except Fusobacterium were found (Li et   al., 2014 ). In contrast, Italians carried common genera (Neisseria and Prevotella). Li et   al. (2014)  performed a comparative microbiome analysis of Alaskans, Germans, and Africans, including the Democratic Republic of Congo (n = 15), Sierra Leone (n = 13), and Uganda (n = 38) and revealed more similarities between Alaskans and Germans than Africans at the genus and OTU levels (Li et   al., 2014 ). Both native Alaskans and Germans shared 13 common genera, while Alaskans and Africans shared only six genera (Neisseria, Campylobacter, Granulicatella, Megasphaera, Selenomonas, Actinomyces) and Germans and Africans carried three common genera (Actinobacillus, Aggregatibacter, and Capnocytophaga). Also, all of the foregoing populations shared only three genera (Streptococcus, Fusobacterium, and Leptotrichia) in common. Nasidze et   al. (2009a)  reported considerable differences in the diversity of the saliva microbiome between African populations, which were attributed to subsistence and dietary patterns. A study involving Sierra Leone and Congo, which are geographically distant but have similar dietary patterns, showed a higher degree of similarity with each other than with Batwa (Nasidze et   al., 2009a ). Although no significant geographical signature of the salivary microbiome was detected in various populations, a frequency variation in the specific genera was found. For example, significant fluctuations in the frequency of Enterobacter were seen. Enterobacter constitutes approximately 28% of the sequences obtained from the Congo but not California, China, Germany, Poland, or Turkey. Furthermore, Serratia showed a relatively high frequency among Bolivians (Nasidze et   al., 2009a ). Murugesan et   al. (2020)  characterized the salivary microbiome of the Qatari population, which was associated with gender, aging, oral health, 16 smoking status, and coffee or tea consumption. They found that Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria are the common phyla, with Bacteroidetes being the predominant phylum, and at the genus level Prevotella, Porphyromonas, Streptococcus, and Veillonella (mean values of males and females, 54.3%, 8.1%, 6.6%, and 6.22%, respectively) were the most abundant in Qatari saliva samples. These results indicate that Qatar differed from countries where Firmicutes was the dominant phylum such as Bangladesh, UK, Japan, South Korea, and Brazil. Further sampling of various populations is required to demonstrate the unique geographic differences of each region. Vaginal Fluid Identification of vaginal fluid in sexual assault cases is desirable for some forensic investigations. In particular, mixed samples containing vaginal fluid mixed with semen may suggest vaginal intercourse in sexual assault cases (Akutsu et   al., 2012 ). Bacterial markers have been suggested to play a role in vaginal fluid identification based on the presence of Lactobacillus (87.5%), Lachnospiraceae (2.3%), Prevotella (1.1%), Alcaligenaceae (1.0% ), Erysipelatoclostridium (0.9%), Corynebacterium (0.7%), Peptoniphilus (0.6%), Bifidobact erium (0.6%), Anaerococcus (0.5%), and Staphylococcus (0.5%) (Dobay et   al., 2019 ). Also, Subdoligranulum (2.3%), Blautia (1.7%), Escherichia-Shigella (0.5%), Anaerostipes (0 .4%), and Stenotrophomonas (0.3%) were found in the exposed vaginal fluid (Dobay et   al., 2019). Lactobacilli play an important role in protecting the host from the urinary tract and genital infections and in maintaining the vaginal microbial balance; they occur predominantly in the vaginal microenvironment of healthy women (Boris et   al., 1998 ; Mc and Rosenstein, 2000; Witkin et   al., 2007 ). The abundance of Lactobacillus promotes acidic vaginal pH, which is the signature of Lactobacillus colonization, and is attributed primarily to the metabolism of glycogen to lactic acid (Mirmonsef et   al., 2016 ; Das Purkayastha et   al., 2019 ). However, vaginal pH and Lactobacillus diversity and dominance differ with individual lifestyles (Das Purkayastha et   al., 2019 ). Recent studies and the human microbiome project reported nearly 60 vaginal microbiomes including four dominant species in the urinogenital tract: L. crispatus, L. iners, L. gasseri, and L. jensenii (Pavlova et   al., 2002 ; Verhelst et   al., 2004; Kroon et   al., 2018 ). We analyzed seven vaginal fluid samples from four countries listed in FMD (Table   1 ) and 13 samples from 10 countries that are not included in the FMD (Table   2 ). Three body sites related to the vagina were listed in the FMD. Bacteria comprising less than 10% from vaginal 17 and inflammatory diseases such as obesity, diabetes, and cancer (Adlerberth and Wold, 2009; Armougom et   al., 2009 ). The most predominant phyla in the human gut are Bacteroides and/or Firmicutes, constituting more than 80% of the total microbiome (Lay et   al., 2005 ). Dietary changes alter the composition of Bacteroides and Firmicutes (Clarke et   al., 2012 ; Scott et   al., 2013 ). Stool samples including 99 samples from 25 countries listed in the FMD (Table   1 ) and 10 samples from each of the 10 countries not included in the FMD were analyzed (Table   2 ). We divided the stool microbiome data reported in the FMD studies into five groups to facilitate analysis based on region and abundance (Figure   4 ). Bifidobacterium species were predominant in East Asia (Japan, China, Taiwan, and Philippines), Prevotella in South Asia (India, Thailand, and Indonesia), and Bacteroides was the most prevalent in West Europe (Germany, Belgium, UK, Ireland, and Spain), North and South America (USA, Canada, Chile, and Argentina) and Australia. The microbiome composition was inconsistent across African countries including Gambia, Ghana, and Tanzania. In contrast, Koreans carried three phyla, which were predominantly found in stool microbiomes (Bacteroidetes, Firmicutes, and Proteobacteria) and accounted for a mean value of 99% of sequences (Shin et   al., 2016 ). Also, Shin et   al. (2016)  reported that Butyricimonas of the phylum Bacteroidetes was predominant in Seoul (mainland), while Catenibacterium from the Firmicutes phylum was dominant in Jeju (an island). In addition, Japanese, Korean, and Chinese communities carried an abundance of Firmicutes, Actinobacteria, and Bacteroidetes, respectively (Nam et   al., 2011 ). Japanese harbored predominantly Bifidobacterium and Clostridium, whereas Koreans carried Prevotella and Faecalibacterium, and Chinese had Bacteroides in their gastrointestinal microbiomes (Nam   et   al., 2011 ). Zhang et   al. (2015)  reported that Phascolarctobacterium of Firmicutes was the most abundant microbe in Chinese. In an Indian study, Tandon et   al. (2018)  analyzed urban cohorts derived from Western India and reported clear taxonomic differences in the microbiomes of American, Chinese, and Japanese populations. More than 80% of the sequences of gut microbiome derived from stool samples in the western Indian cohort belonged to five genera: Prevotella, Faecalibacterium, Alloprevotella, Roseburia, and Bacteroides (Tandon et   al., 2018 ). These results were in line with FMD data which showed that Prevotella was the most predominant in the Indian urban population. A characteristic gut microbiome profile of the Himalayan population exhibited an increased abundance 20 of Treponema and Prevotella and a decreased abundance of Bacteroides and Bifidobacterium (Jha et   al., 2018 ). The difference across studies in the same country is attributed to sequencing methods, differences in sample preparation, bioinformatics, and cohorts from different ethnicities and provinces, especially in China. The distribution of the microbial community in the stool samples is explained by the composition of the gastrointestinal microbiome. Compared with other samples, the stool samples tend to be affected by the habitat and dietary patterns. Gupta et   al. (2017)  reviewed the variation in the gastrointestinal microbiome based on the degree of urbanization. Models involved hunter-gatherers (Hadza of Tanzania, Pygmies of Central Africa, the Matses of Peru, and Amerindians of Venezuela), traditional farmers and fishermen (Bantus of Africa, the Tunapuco of the Andean highlands, or the rural Malawian communities), and urban industrialized populations (the USA and European) (Gupta et   al., 2017 ). The findings suggest that the higher the transition from hunter-gatherers to urban population, the lower was the microbial diversity along with depletion of Prevotella and proliferation of Bacteroides. From a forensic perspective, access to the gastrointestinal microbiome may provide clues to the whereabouts of suspects based on a comparative analysis of the aforementioned studies involving the composition and diversity of stool samples and microbial clusters. Sequencing Platforms It is important that the sample processing methods, data generation, and data analysis should be standardized for comparing microbiome data. Therefore, future investigations should reduce the differences in methodology and focus on standardizing analyses to ensure a better interpretation of the observed variation in these studies. Two sequencing approaches including 16S rRNA gene sequencing and whole genome shotgun sequencing have been used in the human-associated microbial analysis (Kuczynski et   al., 2012 ). In this section, the strengths and limitations of 16S rRNA gene amplicons and shotgun metagenomics are reviewed. Microbiome analysis based on 16S rRNA gene amplicons entails PCR amplification and sequencing of a variable region (or multiple regions). In contrast, shotgun metagenomics entails fragmentation and amplification of the microbiome genome, followed by sequencing (Ranjan et   al., 2016 ). The primary microbial target is the 16S rRNA gene, which has been widely used. It is appropriate in the human/host DNA background (e.g., a skin swab) because the 16SrRNA primers only amplify the bacteria and archaea domains of life. The PCR step is appropriate for low biomass samples as it is inexpensive. However, single amplicon 21 sequencing is limited by the differences in the relative abundances measured by bacterial community sequencing compared with the true relative abundances, and the primers used for amplification may misalign with the target region in some species. Human microbiome shotgun metagenomics can overcome these limitations, and theoretically, the entire genome of a microbe is amplified due to the additional targets for analysis. Furthermore, no amplification bias is detected since it bypasses primer-dependent PCR amplification. However, shotgun metagenomics entails higher sequencing costs and stochastic effects and generates a large number of uninformative reads that are not variable between taxa, which is a waste of sequencing efforts. In contrast, recently, Schmedes et   al. (2018)  reported the use of hidSkinPlex, a panel of informative targets derived from skin microbiomes, which could be used with machine learning tools in human forensic applications. Clade-specific markers were investigated based on 286 bacterial (and phage) family-, genus-, species-, and subspecies-level markers, which were derived from publicly available datasets generated by human microbiome shotgun metagenomics (Oh et   al., 2016 ). Also, the hidSkinPlex markers were used to classify skin microbiomes including three body sites (foot, hand, and manubrium). Based on supervised learning, all samples were correctly classified and the body site origin was estimated up to 86% accuracy. This preliminary approach yielded additional targets for analysis and interpretation after amplification. Furthermore, they provided insight into the utility of skin microbiome for human identification and may represent an appropriate balance between single-target and shotgun sequencing approaches. Studies continue to examine and analyze samples more precisely. Recent studies have reported microbial community sequencing, including amplicon and shotgun metagenomic sequencing, for vaginal microbiome analysis (Berman et   al., 2020 ). It is important to distinguish the different microbial species, since Lactobacillus and Gardnerella are the predominant genera in most of the vaginal microbiome in normal healthy women, especially when vaginal fluid is used as evidence (Ghemrawi et   al., 2021 ). Likewise, microbial communities in each sample exhibit a characteristic distribution. Ethnic groups can be distinguished at the level of phylum or genus. Therefore, the accurate forensic application requires analysis at the taxonomic level based on the characteristic distribution of the microbial communities in each sample for optimal comparison. Whole microbial genomes obtained via shotgun metagenomic sequencing provide insight into functional genes and pathways of the microbiome, which cannot be obtained via 16S rRNA gene amplicon 22 During the anagen phase, the hair is actively growing, and materials are deposited in the hair shaft by cells found in the follicle. Metabolically active and dividing cells above and around the dermal papilla of the follicle grow upward during this phase, to form the major components of the hair—the medulla, cortex, cuticle, and accompanying root sheath. In the telogen phase, the follicle is dormant or resting. The transition period between the anagen and telogen phases is referred to as the catagen phase Hairs are routinely lost during the telogen phase and often become a primary source of evidentiary material. An example of this natural shedding process can be seen when one combs through the hairs on the head. It is not uncommon for hairs of this type to be transferred to another individual or to an object during physical contact. Hairs can also become dislodged from the body while they are in an actively growing state, such as by pulling or by striking with an object. The microscopical appearance of the root area will allow for the determination of the growth phase.On a healthy head, 80 to 90 percent of the hair follicles are in the anagen phase, 2 percent are in the catagen phase, and 10 to 18 percent are in the telogen phase. Once the hair reaches the telogen phase, the follicles have achieved a mature, stable stage of quiescence. During the telogen phase, the hair is anchored in the follicle only by the root, which is club-shaped. The germ cells below the club-shaped root will give rise to the next generation of an anagen hair. The replacement of human scalp hair occurs in a scattered mosaic fashion with no apparent wave-like or seasonal pattern. The average period of growth for scalp hair is approximately 1,000 days; the resting phase lasts about 100 days. Approximately 10 percent of the hairs on a human head (100/1000), therefore, are in the quiescent telogen phase, and a minimal amount of force—such as that from combing—is required to dislodge the hairs from the dormant follicle. Animal Hairs Animal hairs discovered on items of physical evidence can link a suspect or location to a crime of violence. A victim placed in a vehicle or held at a location where animals are routinely found often results in the transfer of animal hairs to the victim’s clothing. Cat or dog hairs can be found on the adhesive portions of ransom and extortion notes prepared by pet owners. The transfer of pet hairs to the victim or crime scene may also occur when the suspect is a pet owner and has animal hairs on his or her clothing when the contact occurs. This is referred to as a secondary transfer of trace material. When an animal hair is found, it is identified to a particular type of animal and microscopically compared with a known hair sample from either an animal hair reference collection or a specific animal. If the questioned hair exhibits the same microscopic characteristics as the known hairs, it is concluded that the hair is consistent with originating from that animal. It is noted, however, that animal hairs do not possess enough individual microscopic characteristics to be associated with a particular animal to the exclusion of other similar animals. The collection of a suitable known animal hair standard is necessary before a meaningful comparison can be conducted. Because hairs can vary widely in color and length on different areas of the body of an animal, hairs should be collected from each area. While a minimum number of hairs is difficult to determine, good judgment should be used in collecting enough hairs to represent the various types and colors of hairs found on the animal. The sample should contain full-length hairs and should include combings as well as pluckings. If the animal is not available for sample collection, a brush or comb used for the animal may be substituted. Sometimes hair samples collected from a dog or cat bed may be useful when actual samples from the animal cannot be obtained. 25 Human Hairs As stated previously, physical contact may result in the transfer of hairs. These can transfer directly from the region of the body where they are growing—a primary transfer—or they can transfer from the clothing of individuals—a secondary transfer. It has been reported that approximately 100 head hairs are shed by an individual each day. These hairs are shed on clothing and on items in the environment. Contact between a victim and a suspect’s environment can easily cause a secondary transfer of hair. Hairs that are found on the clothing of suspects or victims and appear to have fallen out naturally may be the result of primary or secondary transfer. Hairs that have been forcibly removed may suggest a violent confrontation. Body Area Determination The body area from which a hair originated can be determined by general morphology. Length, shape, size, color, stiffness, curliness, and microscopic appearance all contribute to the determination of body area. Pigmentation and medullar appearance also influence body area identification. Hairs that exhibit microscopic characteristics shared by different anatomical areas are often referred to as body hairs. These include hairs found on the upper legs, lower abdomen, and back. Because there is a wide range of interpersonal variation in head and pubic hairs, the majority of work in forensics has been in comparing and differentiating hairs from the head and pubic regions. Head Hairs Head hairs are usually the longest hairs on the human body. They are characterized as having a uniform diameter and, often, a cut tip. Head hairs can appear uncut, with tapered tips but are more often cut with scissors, razors, or clippers. In general these hairs are subject to more alteration than hairs from other body areas. Alterations to the natural appearance of hair include use of hair dyes, rinses, permanents, frosts, and other chemical applications. Environmental alterations can result from exposure to excessive sunlight, wind, dryness, and other conditions. Because these hairs can be affected by a number of environmental and chemical conditions, it is recommended that head hair samples be obtained as soon as possible from suspects and victims of crime. Head hair samples obtained years after a crime are generally not suitable for meaningful comparison purposes. As head hairs are routinely compared in a forensic laboratory, it is important to obtain suitable known samples from suspects and victims and possibly from other individuals (elimination samples). The known sample should contain a random sampling of hair from different areas of the scalp. The number of hairs required for a meaningful comparison may vary depending on the uniformity of characteristics present in the hairs from an individual. Because this is not known when the hair sample is taken, obtain at least 25 full-length hairs. This hair sample should include both plucked and combed hairs, packaged separately.   Pubic Hairs Pubic hairs are also routinely compared in a forensic laboratory. As with head hairs, considerable variation exists between individuals in the population. Pubic hairs are not subject to as much change as head hairs over time, and because of this, a sample taken a year or more after a crime may still be suitable for meaningful comparison purposes. It is recommended that a known pubic hair sample be obtained as soon as possible after a crime and should contain at least 25 full-length hairs taken from different areas of the pubic region. 26 Pubic hairs are generally coarse and wiry in appearance. They exhibit considerable diameter variation or buckling and often have a continuous to discontinuous medulla. While tapered tips are common, these hairs may also be abraded or cut. Facial Hairs Facial hairs are more commonly called beard hairs or mustache hairs. These hairs are coarse in appearance and can have a triangular cross section. Heavy shouldering or troughs in the hair are observed under magnification. Other characteristics include a wide medulla and a razor-cut tip. The presence of facial hairs on the clothing of a suspect or victim may help establish contact between these individuals. While these hairs may be compared microscopically, the significance of the association may not be as great as head hair and pubic hair associations. Limb Hairs Hairs from the legs and arms constitute limb hairs. These hairs are shorter in length, arc-like in shape, and often abraded or tapered at the tips. The pigment in limb hair is generally granular in appearance, and the medulla is trace to discontinuous. While limb hairs are not routinely compared in a forensic laboratory, they can differ in appearance between individuals. These differences, however, are not considered sufficient to allow limb hairs to be of value for meaningful comparison purposes. The presence of leg or arm hairs on certain items of evidence may help to corroborate other investigative information. Fringe Hairs Hairs originating from areas of the body outside those specifically designated as head or pubic are generally not suitable for significant comparison purposes. These hairs might originate from the neck, sideburns, abdomen, upper leg, and back. Other Body Area Hairs Axillary (underarm) hairs, chest hairs, eye hairs, and nose hairs are not routinely compared. As with limb hairs and fringe hairs, their presence may help to corroborate information obtained during an investigation. Racial Determination A human hair can be associated with a particular racial group based on established models for each group. Forensic examiners differentiate between hairs of Caucasoid (European ancestry), Mongoloid (Asian ancestry), and Negroid (African ancestry) origin, all of which exhibit microscopic characteristics that distinguish one racial group from another. Head hairs are generally considered best for determining race, although hairs from other body areas can be useful. Racial determination from the microscopic examination of head hairs from infants, however, can be difficult, and hairs from individuals of mixed racial ancestry may possess microscopic characteristics attributed to more than one racial group. The identification of race is most useful as an investigative tool, but it can also be an associative tool when an individual’s hairs exhibit unusual racial characteristics. Caucasoid (European) Hairs of Caucasoid or Caucasian origin can be of fine to medium coarseness, are generally straight or wavy in appearance, and exhibit colors ranging from blonde to brown to black. The hair shafts of Caucasian hairs vary from round to oval in cross section and have 27  Research studies have shown that hairs from two individuals are distinguishable; that no accidental or coincidental matches occurred; and that such accidental or coincidental matches would, in actual casework, be a relatively rare event; and  The significance of a hair match is a median point between the above statements. It has also been stated that hair evidence is only of value when used in conjunction with other evidence. Positive hair comparison conclusions may be weakened by the presence of incomplete hairs; by common, featureless hairs; and by known samples with large intrasample variation. Conversely, positive hair comparison conclusions are strengthened by the presence of two or more mutually dissimilar hairs that are similar to a known sample; by hairs with unusual characteristics; by two-way transfers; and by additional examinations of confirmation, such as DNA and sex-typing. Normal negative hair comparison conclusions are weakened by deficiencies in the known hair sample, including too few hairs, unrepresentative hairs, incomplete hairs, and a significant temporal difference between the offense and the collection of the known sample. Negative hair comparison conclusions are also weakened by the presence of incomplete questioned hairs and by similarities and differences within the hair sample. Factors which strengthen normal negative hair comparison conclusions include a large quantity of known sample hairs; little intrasample variation within the known sample; and hairs that are very dissimilar, such as those exhibiting distinct racial and/or microscopic characteristics. Two or more questioned hairs that are found together in a clump and are dissimilar to the known sample will also support a negative hair comparison conclusion. Physical peculiarities: Physical properties are properties that can be measured without changing the identity of the evidence. For example, when forensic scientists calculate the density of glass, they divide the mass of the glass by its volume. Measuring mass and volume does not affect the chemical makeup of the glass. Therefore, density is a physical property. Other physical properties include colour, melting point, boiling point, odour, and viscosity. Changes to substances that do not alter the chemical makeup of the substance—cutting, shredding, melting, or freezing —are physical changes. To make optimum use of physical evidences, Forensic scientists have tried to classify evidences in different ways on the basis of several different perspectives. Some of the 30 classification systems are more useful than others. But none of the systems, however, can incorporate all the perspectives into account individually. At first instance, one might wonder why physical evidences are classified; the following points will provide the answer:  The class or type of evidence can be very important in determining its value  How has it to be collected?  What else needs to be collected along with the samples such as controls and exemplars?  And the most important is what sort of scientific tests should be conducted to draw varied conclusions from them? Legal distinctions among different types of evidence help to determine their admissibility in the court of law. These schemes apply to all evidence, not only scientific or technical evidences. The following schemes to classify the physical evidences have been proposed by the scientists, which are comparatively more realistic than others: 1 General nature of the evidence 2 Type of material 3 The physical state of the evidence 4 The type of crime 5 Types of questions to be resolved 6 According to the way the evidence was produced, and 7 According to the appropriate laboratory approach Every scheme is useful in offering a different conceptual perspective related to physical evidence, but some of them are more useful than others. 1 General Nature of the Evidence: According to the general nature of items, the physical evidence can be classified as physical, chemical, or biological (the biological can be related to human, animal or vegetable). In this scheme, the examples of physical would be a paint, plastic, glass, firearm, cartridge case, tool, tool mark, whereas a drug sample would be chemical. Examples of Biological would include hair, pollen grains, and bloodstains DNA etc. Besides having limited value, this scheme might serve to remind the investigator the type of precaution required to collect and preserve the items of the biological category, which are perishable. 2 Type of Material: This classification system of evidence is based on the type of material of which it is composed of such as paint evidence, blood evidence, wood evidence, metallic evidence, glass evidence, plastic evidence, and paper evidence etc. This scheme is of limited utility because a fingerprint found on glass, e.g. is handled and examined essentially in the same manner as finger print found on any other nonporous surfaces like paint, polished metal, 31 plastic or a glossy photograph. Similarly, in case of a tool marks found on metal has to be examined and compared more or less in the same way as one found on some other type of materials? In these types of cases, the nature of the material itself is not very significant but the way in which something has interacted with the substrate material and produced the evidence. But there are some exceptions which are also worth mentioning. If the nature of the material undergoes alteration with the passage of time, proper precautions should be taken to preserve it, or at least to document. Examples includes a bite marks on skin or perishable food, footprint in snow, tool marks or any other evidence that might evaporate, such as petrol. Individualization of materials can be attained on the basis of subtle differences present in their composition wherever required, almost similar laboratory methods are used for materials that have been grouped together in this classification scheme. Microscopic fragments of glass, for example, would be compared using the same techniques, regardless of the kind of object involved. 3 The Physical State of the Evidence: Evidences, like other matter, can be categorized on the basis of its physical state like solid, liquid, or gas. Example of solidstate category includes most types of evidence encountered cartridge case, firearms, glass, tools, clothing, and paper etc. Fewer items of evidence would be placed in the liquid or gas categories. Important examples of liquid evidences include liquid blood samples (either evidence samples or known controls), alcohol and accelerants collected in connection with the investigation of suspected arson case. Gas samples may occur as evidence more often than one might think, but they are rarely recognized as such and even more rarely collected. Specific types of devices can be used to collect sample of gases and vapours at crime and fire scenes. This classification scheme is not useful in any general way. However, it can serve important role to remind investigators about the requirements to have secure packaging for liquid and gas samples so that they do not get leaked or evaporate from their containers. This simple precaution is often over looked in cases from where volatile residues collected from fire scenes. Accelerant samples are analysed in the laboratory where the chances of getting useful results from an analysis of such samples are remote, if not collected and packed properly. 4 Type of Crime: Another system to classify the physical evidence is based on the type of crime from which it has been collected. Thus, for example, the evidences may be related to assaults, rape, homicide, burglary cases, and so on. This scheme might have value in certain situations, but it should be appreciated that any particular type of physical evidence can be found in connection with the investigation of virtually any kind of crime. Different physical evidence types cannot be restricted to legally defined crime classifications. For example, the blood/blood stain evidence very frequently found in the investigations of assault and homicide, but bloodstains can also be recovered as important evidence at crime scenes related to burglary and other property crimes also. Similarly, tool mark evidence can be recovered from almost all types of serious crimes like homicide; assaults and burglary. Therefore, there is some correlation existing between the type of crime and the type of evidence, but it is not as perfect as it should be. Unexpected and unpredictable types of physical evidence could be the most crucial in a particular investigation, and could be overlooked by an investigator with preconceived notions about what to expect and what is likely to be significant. 5 By the Types of Question to be Resolved: In this classification system, evidences are classified according to whether it will be useful in the reconstruction of the event, by proving an element of the crime, in linking a suspect to a victim or to a crime scene, in excluding or 32