Get it off, but keep it: Efficient cleaning of hair shafts with parallel DNA extraction of the surface stain

Forensic Science International: Genetics 45 (2020) 102210

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Get it off, but keep it: Efficient cleaning of hair shafts with parallel DNA extraction of the surface stain

T

Jana Nauea,b, Timo Sängera, Sabine Lutz-Bonengela,b,* a b

Institute of Forensic Medicine, Medical Center – University of Freiburg, Forensic Molecular Biology, Albertstrasse 9, 79104, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany

A R T I C LE I N FO

A B S T R A C T

Keywords: mtDNA analysis Hair analysis Cleaning methods Sanger sequencing Massive parallel sequencing

The analysis of hair samples is a common task in forensic investigations. Material transferred to the surface of a hair during a crime challenges the analysis as it has to be removed efficiently. However, the removal of the stain can also lead to a loss of information on stain contributors. DNA analysis of the stain itself might thus be helpful for the forensic investigation. The aim of this study was the examination of different methods to remove common biological surface stains completely from human hair shafts without hampering the parallel DNA extraction of the cleaned hair shaft and the isolated surface stain (blood, saliva, vaginal secretion, semen, and skin flocks). Four different methods of cleaning (water, lysis buffer, swabbing, NaClO) were compared to their cleaning efficiency as well as their success of mtDNA analysis of three hair donors and the original five stains on the hair. In order to test the suitability of this procedure for future analysis methods, a selection of samples were also sequenced with MPS. Additionally, nuclear DNA analysis of the stain DNA was performed using a screening STR assay to test the potential success for detection of a STR profile. The most efficient removal of the stain was achieved using NaClO, however compromising further analysis of the stain DNA. The best results for cleaning and parallel stain analysis were obtained using a swab moistened with 0.5 % SDS for surface cleaning. Especially water failed to remove stains efficiently, leading to a high amount of mixed mtDNA in the DNA extracts. MPS showed an increased sensitivity for detection of minute mixtures.

1. Introduction Shed hairs, but also body and pubic hairs are one of the most common types of evidence collected at crime scenes. The analysis of hair is therefore important for forensic investigations [1–9]. Hairs usually contain a low amount of DNA in an often degraded state [10]. While conventional STR analysis can still be successful for hairs with roots, it usually fails when applied to telogenic hair or hair shafts. In these cases, the DNA fragments are too few and sizes too small for routinely employed targeted PCR amplicons [11,12]. The analysis of mitochondrial DNA (mtDNA), although of less discriminatory power than nuclear DNA profiles, is often an alternative due to the high mtDNA copy number per cell (compared to nuclear DNA), and its high resistance to degradation [7,12,13]. In specialized laboratories, mtDNA analyses of hairs show success rates up to 90 % [4–6,13,14] even for hair fragments smaller than 2 mm [15], and for historic cases and “museomics” [13,16–18]. Although hair is assumed to be resistant to

penetration by contaminant DNA and different methods with variable efficiency exist for removal of the stain [7,19–23], the highly sensitive mtDNA analysis can still lead to mixed mtDNA sequences if foreign extracellular mtDNA or biological fluids remain on the hair’s surface after insufficient cleaning. A stain-destructing cleaning procedure has the disadvantage of losing evidence material if the DNA transferred has a relevance to the crime. Thus, the cleaning technique should not only remove all of the non-hair DNA, but also allow for further DNA analysis of the stain removed from the hair. In this study, we artificially stained hair shafts of three donors showing various hair characteristics with five different biological materials (blood, saliva, vaginal secretion, semen, and skin abrasion). To allow an easy implementation into the laboratory workflow it is advantageous to have one cleaning pipeline for removal of different types of staining. This is also important as in forensic cases, multiple types of biological fluids can occur as stain on one single hair, and the type of stain is not necessarily recognizable. Four common cleaning methods

⁎ Corresponding author at: Institute of Forensic Medicine, Medical Center – University of Freiburg, Forensic Molecular Biology, Albertstrasse 9, 79104 Freiburg, Germany. E-mail address: [email protected] (S. Lutz-Bonengel).

https://doi.org/10.1016/j.fsigen.2019.102210 Received 30 September 2019; Received in revised form 20 November 2019; Accepted 25 November 2019 Available online 26 November 2019 1872-4973/ © 2019 Elsevier B.V. All rights reserved.

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2.4. Analysis of mitochondrial DNA

(washing with water, a mild lysis buffer, 3 % NaClO, and mechanical cleaning with a swab moistened with 0.5 % SDS, respectively) were compared regarding their cleaning efficiency and (additional) successful stain analysis. The mtDNA analysis of all samples was performed using Sanger sequencing (SA) and for selected samples by massive parallel sequencing (MPS) due to its higher sensitivity. DNA was extracted from the respective cleaning medium, and a pentaplex STR analysis was performed [24].

2.4.1. Sanger sequencing (SA) Two parts of the mitochondrial control region were amplified allowing the discrimination between all hair and stain donors by at least two positions, except for one hair/stain combination, (cf. Suppl. material S1). Primer pairs L16268/ H00159 and L00314/ H00611 from the ‘Mito-Midi’ mtDNA amplification strategy were used for amplification [26]. Due to a lack in differing positions within the selected regions in the hair/stain combination of IDs 4 and 7, additional analysis using primer pair L15908/ H16432 [26] was required (Suppl. material S2B). PCR and sequencing were performed as described in [27], with small modifications: a duplex PCR reaction was performed using the SA-Advantage Buffer (Takara Bio USA, Mountain View, CA, USA), and an increased reaction volume of 15 μL including 5 μL of DNA extract as template. Sequencing was performed using primers H00159 and L00314, and additionally H16432 for the sample combination ID 4/ ID 7 (cf. Suppl. material S2B). Capillary electrophoresis was run on the 3130xl with Foundation Data collection Software v3.0 (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). The resulting sequences were aligned to the rCRS [28] using Sequencher 5.4.6 software (GeneCodes, Ann Arbor, MI, USA). Re-sequencing of samples was performed in 16 cases, in which initially a partial sequence was determined, not covering the minimal amount of two positions for mixture calling.

2. Materials and methods 2.1. Sample collection and hair shaft staining For the comparison of cleaning methods prior to DNA extraction from hair shafts, hairs were prepared, and stained with biological material (Suppl. material S1): Freshly washed hairs from three donors (ID 3 - ID 5) with different hair characteristics in respect to each other (thick/ black, medium/ dyed, and thin/ grey), as well as skin flocks and body fluids (blood, saliva, vaginal fluid, and semen) from a total of five different donors (ID 6 - ID 10) were collected with informed consent. Hairs of two individuals (ID 1, ID 2) were taken for initial pre-experiments (data not shown). Ethical approval was obtained from the University of Freiburg Ethics Committee (467/18). The study followed the ethical principles stated in the Declaration of Helsinki of the World Medical Association [25]. Donors (ID 3 - ID 10) were selected based on their mtDNA haplotypes (detected by prior mtDNA typing using buccal swab DNA) to ensure a differentiation of all analyzed sequences (Suppl. material S1). In total, 75 hairs from each hair donor were plucked using tweezers (three hair shafts per stain type for a triplicate analysis of single hair shafts, 5 different stain types, four cleaning procedures, and one uncleaned control). Each plucked hair was cut 0.5 cm distal from the root and 1 cm of the hair shaft was used for the experiments. To obtain equally stained hair shafts, 15 hair shafts from each individual were incubated (5 min, 600 rpm at room temperature) in one tube containing freshly taken blood, saliva, semen, and vaginal secretion, respectively. To transfer skin flocks, hairs were rubbed in the skin flake donor’s hands for one minute. After transfer of the biological material, all hair shafts were singularized and air dried over night at room temperature.

2.4.2. Massive parallel sequencing (MPS) 2.4.2.1. PCR, library preparation and massive parallel sequencing. Selected regions of the control region for discrimination of all donors were amplified and sequenced. Amplicons and sequencing lengths were shorter than in SA due to the common MPS approach of 150 bp paired-end-sequencing. Primer sequences were selected from the ‘Mito Mini’ approach [26] adding an Illumina Nextera Transposase Adapter for MPS (Suppl. material S2B). Mitochondrial DNA was amplified using two multiplex amplifications including each two (L16197/ H16433, L16450/ H00052) and three (L16363/ H16509, L16533/ H00180, L00402/ H00599) primer pairs. PCR conditions were as for SA but in a 10-μL volume with 2.5 μl of DNA input. The two multiplex reactions per individual were then pooled (3 μL MMA, 4 μL MMB), cleaned with 1.9x magnetic beads (GE Healthcare, Little Chalfont, UK) prepared according to [29], and eluted in 20 μL water. Depending on the amount of PCR product as verified by gel analysis, 3 μL or 6 μl of pooled sample were added to 12.5 μL Q5 Hot Start Ultra HiFi PCR Master Mix (New England Biolabs, MA, USA), 5 pmol of each index-primer (dual indexing), and water (ad 25 μL). Run conditions were: 98 °C 1 min, 72 °C 5 min; 6 cycles: 98 °C 30 s, 62 °C 2 min, 72 °C 2 min; final elongation at 72 °C for 3 min. PCR products were cleaned twice with prepared 1.6x beads (GE healthcare) and eluted in 20 μL water. DNA quantity of each amplicon pool was measured using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), and amplicon pools were combined in equimolar ratios. A maximum volume of 10 μL was used for each sample. A final 10.5 pM library containing all samples was paired-end sequenced on a MiSeq (Illumina, San Diego, CA, USA) using the Micro 300 bp v2 Kit (Illumina). Samples with low coverage were repeated using a Micro 300 bp v2 kit and Nano 300 bp v2 kit, respectively.

2.2. Cleaning methods Four different methods were used to remove the surface stain from the hair shafts: 1) washing with 500 μL DNA-free water for 5 min at 37 °C, 900 rpm; 2) washing with 500 μL mild lysis buffer “Buffer AL” (Qiagen, Hilden, Germany) for 5 min at 37 °C, 900 rpm; 3) washing with 500 μL 3 % sodium hypochlorite (NaClO, Roth, Karlsruhe, Germany) for 5 min at 37 °C, 900 rpm; 4) wiping with a DNA-free swab (Sarstedt, Nümbrecht, Germany) moistened with 0.5 % SDS. The supernatants/ swabs (hereafter referred to as “cleaning medium”) were collected for DNA extraction. As controls, three hair shafts of every donor/stain combination were left uncleaned. Afterwards, all hair shafts were washed for 5 min at 37 °C, 900 rpm with 500 μL 80 % EtOH followed by 500 μL DNA-free water.

2.4.2.2. Data analysis MPS. Primer sequences from the obtained FastQ files were removed using cutPrimers v1.2, and quality checked using TrimGalore v0.4.3 (including the FastQC package) [30]. PEAR v0.9.10 was used to join the paired-end reads [31]. Alignment to the human mitochondrial reference sequence NC_012920.1 was performed using the bwa mem algorithm from the BWA package v0.7.17 [32]. Coverage per amplicon and nucleotide was extracted using the bamreadcount package v0.8.0 [33]; in the case of a second run of a sample, the read counts were added. A minimum coverage of 200 reads was needed for

2.3. DNA extraction DNA was extracted from hair shafts and the cleaning media with the QIAamp Micro DNA Kit (Qiagen) with modifications (Suppl. material S2A). DNA was also extracted from untreated hair shafts and body fluids of the respective donors to verify mtDNA haplotypes and to exclude tissue-/cell-type specific heteroplasmic positions.

2

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further position analysis.

Table 1 Overview of samples analyzed using Sanger sequencing (SA). The DNA extract of the hair shaft and cleaning medium, respectively, were analyzed for their presence of mtDNA. The optimal result is shown in bold, detecting only the DNA of the hair donor in the hair shaft DNA extract and the mtDNA of the stain donor in the cleaning medium.

2.5. Criteria for mixture interpretation To classify a result as a mixture, at least two differing nucleotide positions (in hair vs. stain donor) were necessary. Partial sequences were also included if they showed at least two differing positions (cf. Supplementary material S3). SA results were visually analyzed without further computation; however, a minor mixture component had to clearly rise above background signals. In MPS results, the threshold of 200 reads had to be met. Also, the minor variant at a mixed position had to be covered at least 30 × . The threshold for reporting a minor variant was set to 2 %. As a 2 % minor variant also had to be detected at least in 30 reads, a total coverage of 1500x was needed at a given position in order to report this sort of low-level mixture.

Detected mtDNA

Hair shaft DNA extract (SA)

Cleaning medium (SA)

Hair Mixture Stain No result Total

122 95 6 2 225

3 24 81 72 180

shaft DNA extracts, 107 cleaning media DNA extracts) were analyzed using MPS to compare the sensitivity of mixture detection (regions: 16222–153, 477–574). All results are listed in Suppl. material S3. Mitochondrial DNA of the body fluids/skin flocks of the donors was analyzed by MPS for exclusion of tissue-specific low-level heteroplasmy. As a result, position 16311C in ID5 was excluded as discriminatory position due to a heteroplasmy (16311Y) in the three donors ID 8, ID 9 and ID 10. Additionally, in all the DNA extracts from the cleaning media, four STR loci were analyzed and compared to the stain DNA.

2.6. Amplification of nuclear DNA A pentaplex PCR (FGA, vWA, D3S1358, TH01 and Amelogenin, primer sequences from [24]) was performed to determine if nuclear DNA was retrieved from the supernatants and swabs, respectively. The following PCR conditions were used for amplification: 11 min at 95 °C; 10 cycles 30 s at 94 °C, 90 s at 61 °C, 90 s at 72 °C; 20 cycles 30 s at 90 °C, 90 s at 60 °C, 90 s at 72 °C; 30 min at 68 °C. PCR products were detected on the 3500 Genetic Analyzer with Foundation Data collection Software v1.0 and analyzed using GeneMapper ID-X 1.3 (LT).

3.2. Evaluation of the cleaning method based on Sanger sequencing

2.7. Data visualization and statistical evaluation

3.2.1. Stain removal and determination of hair mtDNA In total, using SA, only the hair’s donor mtDNA was detected in the hair shaft DNA extracts in 54 % of cases, a mixture in 42 %, only the stain mtDNA in 3 %, and no result was obtained in 1 % of the samples (Table 1). Particularly problematic is the detection of only stain mtDNA in the DNA extract of the hair shaft extraction, what we observed in six cases. Four of these observations related to (the analysis of) uncleaned hair shafts, highlighting the essential need of a cleaning step prior to hair lysis. Otherwise the detected mtDNA haplotype could be misinterpreted as originating from the hair and not be connected to the transferred mtDNA. The detection of hair mtDNA as sole mtDNA source or within a mixture (3 and 24 times, respectively) in the cleaning media points to the possibility that the cleaning procedure can lead to a partial loss of hair DNA into the cleaning medium. It should however be considered, that although using freshly shampooed hair, a residual amount of DNA (from skin cells, sebum) of the hair donor might be present on the hair shafts analyzed. Considering all the different stain types, a statistically significant difference of the cleaning efficiency compared to water was calculated for the lysis buffer (p = 0.005), the swabbing (p = 3.1*10−9) and the use of NaClO (p = 2.8*10-8) (Fig. 1). In contrast, no statistically significant difference was found for “no cleaning” vs. “cleaning with water”. The highest cleaning efficiency was achieved with 3 % NaClO (96 %, 43/45 hair shafts showed only hair mtDNA; two hair shaft extractions failed) followed by swabbing (93 %, 42/45 only hair mtDNA; three mixtures). With the other two methods, efficient removal of the stain was only partially possible. In the case of the lysis buffer, 51 % (23/45) of the DNA extracts from the hair shafts still showed a mixture of hair and stain mtDNA despite of cleaning. The water protocol used in this study was not efficient for stain removal from the hair shaft as a mixture was detected in 80 % (36/45) of samples. Nevertheless, optimized protocols based on water in other laboratories might have a higher efficiency for removal of material transferred to hair shaft. In two samples, which were cleaned with water and lysis buffer, respectively only the stain mtDNA was detected, leading to false haplotype determination of the hair. Again, this highlights the issue of a possible wrong interpretation as mentioned above for the four uncleaned hair shafts. For the other uncleaned, stained hair shafts, we obtained only

Visualization and further analysis were performed within Jupyterlab 0.35.4 of the Anaconda Navigator package using Python 3.5, and the packages pandas v0.24.0, seaborn v0.9.0 [34] and scipy v1.2.2 [35]. Statistical analysis was performed to compare the cleaning methods for the hair shaft DNA extract as well as the cleaning medium DNA extract. For initial analysis of differences between the cleaning methods, the non-parametrical Friedman test was used for comparison of all methods. Afterwards, the non-parametrical Wilcoxon test was used for analysis of each cleaning method to water as default method. The significance level was adjusted after Bonferroni correction for multiple testing. A coding for the detected mtDNA results (hair, mixture, stain, no result) was performed scaling the results according to the desired outcome with the opposite as most critical result (hair shaft DNA extract: hair-1, mixture-2, no result-3, stain-4; cleaning medium DNA extract: stain-1, mixture-2, no result-3, hair-4). The Exact Fisher’s Test for m*n tables (Fisher-Freeman-Halton test) for evaluation of differences between the cleaning methods obtaining the desired (only source mtDNA or source mtDNA with minor background) complete or partial sequences was done using SPSS v25 (IBM, NY, USA). 3. Results and discussion 3.1. Overview of analyzed samples and regions The collected 75 hair shafts per person were stained with blood, saliva, vaginal fluid, semen, and skin flocks, respectively to test the stain removal efficiency of four cleaning methods (water, lysis buffer (“Buffer AL”), 3 % NaClO, swabbing), and a non-cleaning control. Altogether, 225 extracts from hair shafts and 180 extracts from the cleaning media (no extract from the non-cleaning control) were analyzed by Sanger sequencing (SA). The regions 16288-135 and 334–590 were considered for mixture resolution/detection. The region 15927–16412 was additionally analyzed for the combination of ID4/ ID7 to determine at least two positions differing between the individuals. From a pre-experiment we concluded that it is sufficient to use freshly shampooed hair to obtain clean hair from the donors as starting material (ID 1, ID 2, data not shown). 203 samples (96 hair 3

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Fig. 1. Number of detected mtDNA haplotypes (only mtDNA from hair donor, mixture, only mtDNA from stain donor) in the DNA extract from the hair shaft. DNA was extracted from the hair shaft (previously cleaned either with water, lysis buffer, NaClO, swabs, or not cleaned). The obtained mtDNA haplotypes were compared to the reference sequences and grouped according to their origin (hair, stain). A result is defined as mixture if at least two positions in the chromatogram showed a mixed peak, which originated from different nucleotides of hair and stain donor at a given position. Additional classification/ grouping of the results with regard to the (type of) stains is shown in Fig. 3. ** p < 0.01, ***p < 0.001 (Wilcoxon test).

Fig. 2. Number of detected mtDNA haplotypes (only mtDNA from hair donor, mixture, only mtDNA from stain donor) in the DNA extract of the cleaning medium. DNA was extracted from the cleaning medium or swab used for cleaning. The obtained mtDNA haplotypes were compared to the reference sequences and grouped according to their origin (hair, stain). A result is defined as mixture if at least two positions in the chromatogram showed a mixed peak, which originated from different nucleotides of hair and stain donor at a given position. Additional separation of the results per stain can be seen in Fig. 3. ***p < 0.001 (Wilcoxon test).

buffer or swabbing, the success depended on the type of the biological stain resulting in a mixed mtDNA haplotype or the haplotype of the stain donor in most (of the) cases (see below). As for the hair, the coverage of all desired positions is advantageous for the evaluation of stain mtDNA. The stain as a sole or major component was detected especially in samples treated by swabbing (29 samples) or lysis buffer (26 samples), respectively. Comparing the cleaning media of all methods, there was no statistically significant difference for the detection rates of complete vs. incomplete/partial sequencing results. Three-quarter of the analyses yielded complete sequences (water (73 %), lysis buffer (74 %), swabbing (73 %)).

hair mtDNA in 28 % (12/45) and mtDNA mixtures in 64 % (29/45) of the samples. Another criterion to consider is the integrity of the hair DNA after the cleaning procedure as especially the incubation with NaClO might lead to DNA degradation due to the strong chemical. A more aggressive lysis buffer than the lysis “Buffer AL” used here can also result in a loss of DNA into the cleaning medium due to premature lysis of the hair shaft during the washing process. Initial experiments prior to the study showed that e.g. the more aggressive “Buffer ATL” also available in the QIAamp Micro kit for tissue lysis led to a prior release of hair DNA into the cleaning medium and therefore to a loss of DNA (data not shown). As the hairs were cut for the experiments, an “open” surface may also have served as a “portal of entry” of the chemicals into the hair. Using NaClO, four samples yielded either no result or a partial sequence (cf. Suppl. material S3). This might however also be caused by a stochastically low mtDNA amount in the respective hair shafts as another study did not find any negative effect on the quantity or quality of the DNA extracted from hair shafts by using 3 % NaClO with a five minute incubation time for cleaning [20]. Higher concentrated NaClO or longer incubation time led in contrast to a decreased mtDNA recovery from the hair [20]. However, it cannot be excluded that the effect of NaClO is hair type dependent and that even our protocol with 3 % NaClO and 5 min incubation time caused damages to the hair shaft, and embedded DNA. The quality of the results, i.e. detection of either complete or partial sequences of the expected mtDNA, was not related to the respective cleaning method; statistical significances were not observed.

3.2.3. Influence of the stain type The study demonstrates that the efficiency of stain removal and the success rate of stain mtDNA detection depend on the stain type. Vaginal fluid and semen showed to be the most “resistant” stains, whereas the removal of skin flocks and saliva is easily possible (Fig. 3). For skin flocks, it has to be considered that their heterogeneous nature as well as their non-homogenous transfer can lead to stochastic effects in cell count, DNA amount, and therefore success of DNA retrieval. These variations can also be a reason for a better result of the uncleaned hair shafts compared to cleaning using water „at random “(Fig. 3). The stain type had no impact on the cleaning efficiency of NaClO or swabbing. In nearly all of the cases, only the hair mtDNA was detected after cleaning. Using the swab, a mixture with the stain as minor component was identified only in three samples (Suppl. material S4A). A high number of mixtures were detected in uncleaned samples, water and the lysis buffer. These were especially observed in extracts from hair shafts that had been stained with vaginal fluid and semen and had been cleaned with lysis buffer. Using water, the stain types blood, saliva, vaginal fluid, and semen led to a high amount of mixtures with different hair-to-stain-DNA ratios (see below). In a few cases, mtDNA from semen and blood were also the only mtDNA haplotype identified in the hair shaft DNA extract. Especially for blood and semen, a high mtDNA amount can be present in the stain covering the hair’s mtDNA. The staining with skin flocks was the least problematic situation, probably due to the low DNA amount transferred to the hair shaft. The high success rate of stain DNA recovery using a swab was independent from the stain type. However, stain type-specific differences were seen for water and lysis buffer-cleaning, as in these cases remains of the stain often stayed on the hair shaft and were not completely transferred into the cleaning medium (e.g. semen and vaginal fluid, cf. Fig. 3). Nevertheless, in the case of a high mtDNA amount in the stain, either the stain itself or a mixture was recovered in the DNA extract of the cleaning medium. One exception was water, in which no saliva was found due to obviously inefficient dissolution. As mentioned before, NaClO cannot be used in cases where the stain in the cleaning medium also has to be analyzed.

3.2.2. Co-extraction of surface stain mtDNA As the biological material transferred to the hair shaft may be related to a crime, the analysis of the cleaning medium to obtain the mtDNA from the stain might be beneficial. In 81 of the DNA extracts from the cleaning media, solely the stain’s mtDNA haplotype was detected after cleaning: In three of the DNA extracts, there was only mtDNA of the hair, and in 24 of the DNA extracts, there were mixtures of both the hair’s and the stain’s mtDNA. No result was obtained in 72 cases (cf. Table 1). The analysis was most successful for mechanical cleaning using a swab (Fig. 2). No mtDNA was detected in the 45 DNA extracts from the NaClO cleaning media using SA. None of the 17 samples selected for verification by MPS (see below) yielded any result (with this method). This outcome was to be expected due to the DNA degradation characteristics of NaClO. The degrading effect of NaClO might even continue during/after the cell lysis of the stain material, which set free the DNA, making it susceptible for the NaClO. An alternative approach might be the removal of NaClO after cleaning and before the lysis e.g. by a column-based clean-up step prior to DNA extraction. Though, it can be expected that most of the DNA would be lost during this pre-processing step. In the case of cleaning using water, lysis 4

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Fig. 3. Mitochondrial DNA Sequencing results from DNA extracts of the hair and cleaning medium in dependence on the biological material transferred to the hair shaft. The left column shows the results obtained for the hair shaft DNA extract in contrast to the detected mtDNA in the cleaning medium DNA extract in the right column. Each row represents one cleaning method. The best results for all different types of stain for hair and stain mtDNA detection in the hair shaft DNA extract and cleaning medium, respectively were obtained using a swab for cleaning.

mtDNA haplotypes as well as their hair characteristics. This resulted in the analysis of thick dark hair, medium dyed hair, and thin grey hair in relation to each other. As one individual per hair type and only three hairs for each condition were used in this study, this study can only provide a first insight into the (potential) influence of the hair characteristics. In general, different mtDNA amounts within the hair shafts can have an influence on the obtained results: the more mtDNA is present in the hair shaft, the higher the chances for successful detection in contrast to the stain. In contrast, the mtDNA of the stain can completely cover the mtDNA of a hair shaft with a low mtDNA amount. The application of NaClO might lead to early degradation of a hair’s DNA if the treatment damages the hair shaft prior to lysis. Using the classification of the results by SA in the uncleaned hair shafts, we could determine how efficient the biological materials were transferred to the hair shafts initially (Supplementary Material S4B, mtDNA haplotype of hair shaft DNA extract, figure with “no cleaning” results). The analysis of the stained hair shafts without any cleaning procedure showed the least amount of stain mtDNA in the hair shaft DNA extract from hair

The differentiation of an additional sequence can be possible if it is represented as minor component in the SA chromatogram. Only a semiquantitative analysis was possible for SA. Mixtures were classified by differentiating a minor component of unexpected mtDNA (i.e. small amounts of stain mtDNA in the hair shaft DNA extract or small amounts of hair shaft mtDNA in the stain DNA extract), balanced detection of hair and stain DNA, and a major component of unexpected DNA (Suppl. material S4A). That analysis showed that if skin flock mtDNA was detected in the hair shaft DNA extract, it appeared only as a minor component. Saliva was also mainly detected as a minor component whereas blood, vaginal fluid and semen were often detected as major components covering the hair’s mtDNA. In contrast hair mtDNA was especially detected in the DNA extracts from lysis buffer (Suppl. material S4A). This might be due to a starting lysis of the hair shaft during the wash-incubation step in the lysis buffer.

3.2.4. Influence of the hair characteristics Three individuals were selected as hair donors based on their 5

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cases – to additional mixture detection. Next to the generally higher sensitivity of MPS for mixture detection, amplification of smaller fragments by MPS probably also allowed the successful amplification of more degraded DNA. In five samples showing a sequence in SA, no result was obtained by MPS, which might have been caused by stochastic effects and by the analytical/quality threshold settings chosen for the MPS analysis. Although equimolarity was pursued for the MPS runs, some samples were underrepresented due to low mtDNA amplicon amount.

donor ID3. This might be caused by a higher amount of hair mtDNA superimposing the stain mtDNA in the sequences or that the different stain materials did not adhere as well to this individual’s hair shaft than to the other participants’ hair. The hair shafts of ID4 and ID5 did not show any difference in stain adherence. In general, blood, semen and vaginal fluid showed the highest “staining power” over all hair types or contained more DNA, which was also deduced from the results of uncleaned hair shafts. Considering the different cleaning methods, DNA extracts from the hair shafts of ID3 showed the least amounts of stain mtDNA. In contrast, if hair mtDNA was detected in DNA extracts from the cleaning media, this mostly referred to hair samples from ID 3 (Suppl. material S4B). Certainly; more individuals have to be analyzed for a deeper insight into the effects of hair structure and mtDNA content. Additionally, quantification of the mtDNA copy number would lead to further information about the influence of the mtDNA amount of the hair as well as the biological material itself.

3.4. Opportunities and limitations of the sequencing methods Both sequencing methods appeared sensitive and led to successful amplification of hair mtDNA, and the stain mtDNA after cleaning, except for NaClO treated samples. SA does not represent an accurate quantitative method for mixture determination as multiple factors affect the representation of mixed sequences in different ways at different positions [36]. Therefore, mixtures were only classified (semi-quantitatively) in the categories minor/balanced/major mixture. As only parts of the control region were analyzed, it cannot be excluded that other positions would have been more appropriate for mixture detection, as mixture detection (strongly) depends on clearly visible mixed peaks/ signals. MPS represents a quantitative method with higher sensitivity of mixture detection, however low-level mixtures (< 2 %, or read count < 30 reads for the minor component), might have been missed. In both cases, interpretation of the major mtDNA component would still be possible, allowing haplotype determination. The Midi-technique applied in SA (amplicon lengths of around 350 bp) is a common approach and was selected for the analysis compared to the Mini-technique with shorter amplicons used for MPS. Compared to MPS, the distinction of spurious background amplifications is more difficult in SA (no separate reads). Therefore, we chose Midi-amplicon strategy with longer PCR fragments for SA sequencing. SA failed in eleven samples compared to MPS, and a result was obtained in four cases where MPS had failed. An advantage of the MPS over the SA was the higher number of positions covered, as at least two positions for differentiation of the individuals needed to be analyzed of the sequence to consider the analysis as a positive result. For all NaClO-cleaned samples, MPS of the cleaning medium DNA extract showed spurious reads below the chosen threshold. Higher DNA input in case of MPS and the use of the Miniapproach in Sanger sequencing [26] might lead to more successful sequencing analysis, but would need to be differentiated from stochastically amplified background. The study was limited to single-source staining. In a forensic case with multiple stain contributors, the DNA obtained from the stain would also be detected as mixed sequence. Depending on the sequence differences between the involved individuals, MPS could help in dissolving this mixture. Another point to consider is the occurrence of heteroplasmy. The body fluids were analyzed for exclusion of tissue-specific heteroplasmic nucleotide positions at the regions of interest, leading to the exclusion of position 16311Y. However, heteroplasmy in hairs is commonly detected, especially when using MPS [37]. It can be heterogeneous particularly between hairs [38]. In our study, we detected one mixed position that could not be explained by either donor or lab staff. It can be hypothesized that this case represents an additional heteroplasmic position observed in a single hair shaft of the respective donor. As a restriction, mixed positions would have covered any additional heteroplasmy. This highlights the importance of co-analysis of hair and stain for interpretation, especially in the case of an unknown donor. The investigation of the cleaning success depends on the sensitivity of the chosen method and thresholds. It should be mentioned that the study concentrates on the removal of material transferred to the hair shaft prior to DNA analysis. In respect to e.g. toxicological analyses, other decontamination techniques (would) have to be considered [39]. Cleaning methods that seemed efficient in a study using a defined protocol can still be found to be inappropriate when using a more

3.3. MPS mtDNA analysis The samples analyzed by MPS were selected according to the SA results. DNA from a minor contributor can be hidden in the background of the chromatogram. Thus, a “single-source” DNA extract can still contain a low amount of “unexpected” mtDNA. Therefore, the following samples were additionally analyzed by MPS: (1) A part of DNA extracts classified as single-source samples by SA to check for low-level mixtures; (2) DNA extracts without any results in SA - here, MPS was performed at least for one of the triplicates. (3) Several mixed DNA extracts were sequenced as controls to compare the SA and MPS results (cf. Suppl. material S3). Thus, the MPS analysis in this study was selective, especially including unexpected results (e.g. samples showing only stain mtDNA in the hair shaft DNA extract or samples without any results), and low-ratio mixtures. A total of 203 DNA extracts were analyzed by MPS. 96 of these samples were extracts from hair shafts and 107 were extracts from cleaning media (Suppl. material S5A). All results are summarized in Suppl. material S3 including the quantification of the mixture ratios and assessment of sequence quality. As described in the material and method section at least two differing nucleotide positions had to be covered in good quality for source differentiation (> 200 reads, minor component with at least 30 reads to be considered a mixture, with a minor nucleotide ratio of 2 %). “No result” was assigned if all amplicons of a sample resulted in a coverage < 200x or with less than two positions covered for source identification. Samples with a low background between 1–2 % at some nucleotide positions were considered as single sequence of the hair and stain mtDNA, respectively, neglecting any background signals lower than the reporting threshold of 2 %. Among the re-analyzed hair shaft samples using MPS, twelve additional mtDNA mixtures in the hair shaft DNA extract were detected. Seven of these samples were classified as hair mtDNA and five as stain mtDNA only using SA (Suppl. material S5A). The results can be explained by the possibility of a more sensitive mixture detection using MPS. Six samples showed in MPS a very minor component (2 % - 5 %) and six samples mixtures that can still be missed using SA (7 % - 20 %). In one of the hair shaft DNA extracts, SA revealed a mixture of hair mtDNA and a minute component of stain mtDNA. This mixture was not detected in MPS, probably due to stochastic effects. Especially in MPS analysis of the selected DNA extracts from the cleaning media, more mixtures were detected (47 instead of ten) (cf. Suppl. material 5B). As for the hair shaft DNA extracts, mainly mixtures with hair mtDNA as minor component (lower than 20 %) were detected. In three cases, mixture components higher than 20 % (24 % - 57 %) were unexpectedly not determined by SA, which was caused by the chosen threshold that a mixed sample was only assigned if at least two mixed (nucleotide) positions were detected. The high discrepancy is also caused by the selection of samples without any result in SA. The more sensitive MPS not only led to successful analysis but also – in some 6

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analysis. The results of the STR pentaplex mostly reflected those of the mtDNA-SA analysis of the cleaning medium DNA extract: in 71 cases neither SA nor STR yielded any result, and in 45 cases both techniques produced a complete STR profile and mtDNA sequence of the stain. As for mtDNA, the success rate also depended on the type of biological stain, with nearly no result for skin flocks and a high success rate for vaginal fluid and semen - especially after cleaning with a lysis buffer or a swab.

sensitive DNA detection method or a slightly different protocol. Therefore a direct comparison to other, especially older studies for cleaning efficiencies is often restricted to certain aspects. Jehaes et al. (2008) observed good cleaning results with various methods, including a differential lysis buffer (without DTT) in contrast to physiological salt solution [21], and Wilson et al. (1995b) demonstrated the superiority of the detergent Terg-a-zyme and ultrasonic treatment in contrast to the use of ethanol for hair cleaning [7]. In contrast, in our study, mild lysis buffer was not as efficient resulting often in mixed sequences. These results might be explainable with an increased detection sensitivity of current SA and MPS technologies. However, also the type of body fluid, the degree of staining, and chemical differences of the buffer/ detergent composition can lead to different study results.

4. Conclusions Cleaning of a hair shaft prior to DNA analysis, especially of mtDNA, is crucial to avoid misleading detection of potential stain DNA. In the extreme case, a wrong interpretation is possible due to misleading results. Forensic laboratories should evaluate the efficiency of their cleaning protocols for proper interpretation of the results and to avoid the observed pitfalls. Often, mixtures/mixed results can be avoided by an adequate cleaning strategy, increasing the evidential value of the hair shaft analysis. With the advent of MPS, the detection of low-ratio mixtures increased due to the method’s higher sensitivity. The additional analysis of the stain can be important in respect to the related forensic case and be of evidential value for the interpretation of the DNA profile obtained. Removing the stain by swabbing was not only efficient but also allowed further DNA analysis of the stain removed from the hair shaft. However, the mechanical removal is laborious and also more complicated for short hair shafts. In these cases, NaClO is a very effective alternative cleaning method with the restriction that any further analysis of the stain is not possible. Initial cleaning of the hair shaft with a swab (for stain DNA analysis) followed by NaClO cleaning (for hair DNA analysis) would combine the advantages of both methods.

3.5. STR analysis As nuclear DNA from the stain was co-extracted with mtDNA from the cleaning medium, we also tried to obtain a STR profile from the stains. Four STR loci and Amelogenin were amplified resulting in a different number of detected alleles dependent on the heterozygosity/ homozygosity status of a STR system (skin flocks, blood: 7 alleles; saliva, semen: 8 alleles; vaginal fluid: 6 alleles). The results were classified in full, no, or partial (more than three alleles) profiles (Fig. 4). In some cases also discrete alleles from the hair shaft donor were obtained, probably due to small nuclear DNA fragments present in hair shafts [12]. Cleaning with NaClO did not yield any interpretable STR results, which is concordant with the mtDNA results. A full profile was obtained in nearly 2/3 of cleaning media DNA extracts analyzed from the swab. Especially for water, a high dropout frequency was observed. Lysisbuffer cleaning resulted on one hand in 40 % of full profiles, but on the other hand also to a high drop-out rate and therefore no measured STR profile. As for mtDNA, no STR analysis was possible for uncleaned samples as no cleaning medium was available. As expected, mtDNA analysis had a higher success rate in determining a profile. No STR results were found in 39 DNA extracts in which mtDNA of the stain or an mtDNA mixture was found. Of these 39 cases, the higher mtDNA detection sensitivity was obtained 13x by the water cleaning, 17x using the lysis buffer and 9x using the swab. Interestingly, in one case with a full pentaplex profile, mtDNA analysis by SA failed. MPS analysis of this sample revealed a mixture with a minor component of 9 %. On one hand, this result could be due to the higher amplicon size in SA compared to mtDNA-MPS and the STR pentaplex. On the other hand, MPS is a highly sensitive technique, which could easily explain the detection of a mixture missed in the STR

Funding Financial support Gesellschaft Freiburg.

was

obtained

from

the

Wissenschaftliche

Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgement We would like to thank all participants of the study and Peter

Fig. 4. STR profiles of the stain obtained from the cleaning medium DNA extract using a pentaplex assay. For a partial profile at least 4 alleles that can be related to the stain (excluding the amelogenin results) were needed to be counted as partial. Shared alleles between hair donor and stain donor are considered as alleles from the stain DNA since only the hair shaft was used initially. Full profile is defined by the total number of different alleles for each stain: skin flocks and blood = 7, saliva and semen = 8, vaginal fluid = 6. 7

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Wiegand from the Institute of Forensic Medicine Ulm for the suggestion to use swabs for the cleaning, as well as reviewer 2 for the suggestion to mention the combination of two methods. We also thank Ulrike Schmidt for commenting on the draft as well as Dirk Lebrecht and Christina Jäger from the Molecular Diagnostics, Pediatric Hematology and Oncology of the University Children’s Hospital Freiburg allocating the MiSeq (Illumina) and their support.

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