A study of old Serbian skeletal remains using ForenSeq DNA Signature™ kit

Journal Pre-proof A Study of old Serbian Skeletal Remains using ForenSeq DNA SignatureTM Kit Eida Almohammed

PII:

S1875-1768(19)30266-5

DOI:

https://doi.org/10.1016/j.fsigss.2019.11.010

Reference:

FSIGSS 1847

To appear in:

Forensic Science International: Genetics Supplement Series

Received Date:

19 August 2019

Revised Date:

11 November 2019

Accepted Date:

11 November 2019

Please cite this article as: Almohammed E, A Study of old Serbian Skeletal Remains using ForenSeq DNA SignatureTM Kit, Forensic Science International: Genetics Supplement Series (2019), doi: https://doi.org/10.1016/j.fsigss.2019.11.010

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Page 1 of 9 A Study of old Serbian Skeletal Remains using ForenSeq DNA Signature™ Kit Eida Almohammed Ministry of Interior- Qatar State [email protected]

Abstract Recent advances in massively parallel sequencing (MPS) has become a very promising technology for massive genetic sequencing [1]. In this study Illumina ForenSeq™ DNA Signature Prep kit was tested to determine if MPS offers a more comprehensive evaluation of degraded samples than the traditional fragment analysis/capillary electrophoresis based method [2]. The use of MPS would therefore reduce the analysis time and augment the identification of human remains. In this context we aimed to analyse the hard tissue degraded samples using ForenSeq™ DNA Signature kit. These samples had given partial profiles with dropout at several loci with GlobalFiler™ kit previously. The MPS kit showed that it is highly sensitive, aids in higher allele recovery for STR loci and provides valuable information about biogeographic ancestry, identity and phenotypic features from a single analysis. The work resulted in highly successful amplification and sequencing of 30 degraded bone/teeth samples using MPS method.

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1.Introduction Recent advances in massively parallel sequencing (MPS), provide advantages to analyse DNA from skeletal human remains, particularly old ones due to enhanced sensitivity and as many more markers can be analysed simultaneously. The ForenSeq™ DNA Signature kit, includes autosomal STRs, Y STRs, X STRs, Identity SNPs and Ancestry SNPs [1]. In the forensic context, the major advantage of these markers is their possible usage with highly degraded DNA as in disaster victim identification and forensic samples [3], [9]. The greatest challenge faced for the analysis of degraded samples, especially for ancient specimens, is the potential for any extracted DNA to have come from a source other than the target [7], [12]. The larger loci (>200bp) are therefore the first to be affected, and are commonly lost [1], [11], [12]. Recent advances in (MPS), provides some advantages to analyse DNA from human remains [7], [14]. An advantage of MPS technology is that shorter STR markers suitable for profiling degraded DNA and SNPs can be multiplexed together. One of the significant advantages to using MPS for forensic analysis is that rather than a profile based on fragment size analysis, the actual sequence of the STR is obtained. Numerous isoalleles can provide the capability to distinguish between two individuals with the same allele call for a locus, thus increasing the discriminatory value of the allele and also aiding the deconvolution of mixtures [5], [13], [14], [20]. In this work we have investigated the performance of the ForenSeq™ DNA Signature kit, on a set of degraded skeletal DNA samples from Serbia.

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2.Materials and Methods 2.1 Typing Success on (Bones samples)

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A number of 30 challenged samples were chosen based on data generated for these samples with standard CE methodology. These samples included DNA from aged bone samples and poor STR results and 30 bone DNA samples that yielded a varying number of typable alleles with CE technology. 2.2 ForenSeq DNA Signature Prep Kit (Beta version)

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The beta version of the ForenSeq DNA Signature Prep kit provides PCR primer mixes for the targeted amplification of 58 STRs, 94 identity informative SNPs (iSNPs), 56 ancestry informative SNPs (aSNPs) and 22 phenotypic informative SNPs (pSNPs) [2]. A primer mix containing a pair of tagged oligos for each target sequence was mixed with the DNA sample. PCR cycles linked the tags to the copies of each target to form DNA templates consisting of the regions of interest flanked by universal primer sequences. The tags were used to attach indexed adapters, which were then amplified using PCR, purified, pooled into a single tube, and then sequenced. This process was done with integrated indexing to support sequencing of 30 samples in a single run including the positive and negative controls. The samples were sequenced on Illumina Miseq FGx [19]. 3.Results

Page 2 of 9 3.1 ForenSeq™ UAS Results

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The ForenSeq™ UAS provided quality and coverage data for all samples and was used to determine STR and SNP genotypes as well as estimate ancestry and phenotype. The analytical threshold was set at 10 reads while the stochastic threshold was set at 30 reads. The ForenSeq™ UAS calculated 1/RMPs from the STR length-genotypes, STR sequence-genotypes, and SNP genotypes. Allele frequencies from the (ForenSeq™) universal software population datasets (African, admixed American, Asian, and European) were used. The ForenSeq™ DNA Signature kit results were quite robust given the nature of the samples. A total of 88.8% of the loci was observed to have amplified, of which 84.3% were <150 bp in fragment size. Of all the markers which amplified the highest proportion present were pSNPs (100%), auSTRs and iSNPs (86.5%) and AISNPs (96.4%). Each sample produced concordant results with the GlobalFiler™ kit. MPS results showed a higher number of allele recovery for autosomal STRs apart from sample No 22 (1406/12). This sample had a DNA quantity of (0.018 ng) and a DI of 14. Sample 11 (90/12) had a high DI 7 and GlobalFiler™ alleles recovered of 40. Sample No 9 had DNA quantity of 0.001ng; DI of 1133 and 33 GlobalFiler™ alleles were recovered. For all the bone samples, the auSTR allele recovery was much higher than the GlobalFiler™ kit despite low quant values and inhibited nature of these samples. Of all the markers amplified the highest proportion present were (100%) phenotypic SNPs, (98.6%) for autosomal STRs, X-STRs, (98.6%) identity SNPs and ancestry SNPs (Fig 1 A&B).

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The discriminatory power for each analysis of degraded DNA from bones, random match probabilities were calculated. The inverse of RMPs (1/RMPs) were reported to give the likelihood of obtaining the same DNA profile from an unrelated individual of the US Caucasian/European population group (Fig 3). The GlobalFilerTM kit was able to consistently achieve the highest discriminating power, having an average 1/RMP value of one in (10 27). The STR sequence-genotype was more discriminating compared to the STR length-genotype because the underlying sequence variation of the STRs increased allelic diversity and therefore power. In addition, the small number of reported loci combined with the low discriminating power of bi-allelic SNPs suppressed the 1/RMP values for the MiSeq® iSNP which had an average 1/RMP value of one in (1012). Looking at the unknown bone samples, the results generated the highest 1/RMP values, an average of one in (1036). Predications were made using the ForenSeq™ DNA Signature kit examined 56 SNPs that estimated biogeographical ancestry of the samples based on principle component analysis (PCA). The ForenSeq™ UAS created PCA plots that reflected the best fit population estimate of each sample’s biogeographical ancestry using 1000 Genomes data: Admixed American, African, East Asian, European. Results showed that the major population of biogeographical ancestry for the skeletal bones was European, which was consistent with antemortem records (Fig 3). The MiSeq® FGx™ platform also had to the potential to estimate hair and eye colour with 22 phenotype informative SNPs [16], [18]. The ForenSeq™ UAS could generate individual probabilities for four hair color categories (black, brown, blonde, and red) and three eye color categories (brown, blue, and intermediate). In order to make these estimates, all phenotype SNP loci had to be detected [6].

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4.Discussion

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For the majority of sequencing work, depth of coverage refers to the average coverage per base across a run, whereas targeted sequencing for forensic applications relies on the actual amplicon or base count for comparison between alleles/loci and for the accurate identification of the present variants. In our study, the depth of coverage for the degraded bone samples found at 95.6% and the range of read depth was 20000 to 4000000 reads, which means that the MPS based analysis were highly sensitive. In this study, for all 30 bone samples, the STR allele recovery using MPS was higher than the GlobalFilerTM kit. Positive and negative controls performed as expected. The results were highly informative and the study demonstrates the potential of MPS to analyze unidentified human skeletal remains and to provide more genetic information from the same initial quantities of DNA sample as that of CE-based analyses. Combined results for all MPS panels included genetic data for 24/24 Y-STRs, 56/56 ancestry informative SNPs, 24/24 phenotype-informative SNPs, 27/27 autosomal STRs (plus amelogenin), and 8/8 X-STRs. The average number of STRs loci detected is (2069) and SNPs alleles (4546). Fig 2B shows that the number of loci which is detected in ForenSeqTM DNA signature kit is more than the normal amplification GlobalFilerTM kit (Fig 2A). An example of old sample which showed a highly degraded sample in Case 627-91, 28 years old gave a partial degraded in GlobalFilerTM EPG (Fig 1B), however, the ForenSeqTM DNA signature kit. It showed a

Page 3 of 9 successful full profile for 27 STRs, 21 Y-STRs belongs to (I2a1b3) Haplogroup of Western Europe predicted in NevGen online tool.

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Ancestry-informative SNP results were obtained for all ancestry SNP markers for 20 samples, 10 samples had a range of 31 to 54 ancestry SNPS respectively. Using the ancestry-informative SNP data, the major population bioancestry bone samples was determined to be Western European Ancestry (86% of samples), followed by Admixed American (7%), and East Asia (7%) since the bone cases were from Belgrade Serbia. The phenotypic SNPs provided blue eye colour predictions and the most likely hair colour was blond or dark blond/brown for majority of the samples in line with the expected phenotypic features [4], [17]. The Nevgen provided Y-Haplogroup predictions that were used. The bone samples from Serbia showed that the samples ancestry predicted to be Western European (86%), Northern and Western European (86%), followed by Admixed American and Europe (7%). The data show that haplogroup I2a was the most common Y-haplogroup in the Europeans studied here. This was reflected in this study where 44% of the bone samples were predicted to belong to haplogroup I2a (44%) followed by E1b1b (17%), G2a (11%), R1b (11%), J2a (6%), C2b1a (5%) and N2 (6%). These Haplogroups are presented in high percentages within European populations, especially in Serbia. A study by Reguiero et al. (2012) indicated that about 58% of Serbian Y-haplogroups (I1, I2a and R1a1a) belong to ancestries and assumed to be preNeolithic [15]. Haplogroup I2a seems to have come out of the Neolithic period and strongly linked to Neolithic cultures in South-East and North-Western Europe.

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It has been recommended to generate data using the limited 150 base single read sequences as it is available now on the MiSeq platform [8], [10], [20]. In this study, 30 Bone samples DNA yielded positive results after sequencing for approximately 230 markers. There are 30 bone samples, which give positive results produced STR profiles that were 98.6% percentage complete (a total of 98.6% loci per full profile) and an of these produced full SNP profiles. The results produced STR loci recovery of alleles generated in MiSeq FGX is higher than the GlobalFilerTM. Of all the markers amplified the highest proportion present were (100%) phenotypic SNPs, (98.6%) for autosomal STRs, X-STRs, (98.6%) identity SNPs, and ancestry SNP.

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Ancestry-informative SNP results were obtained for 54 of the 54 SNP markers amplified via the ForenSeq™ DNA Signature Prep Kit for 20 samples, 10 samples have a range of 31 to 54 ancestry SNPS respectively. About 96.3% of ancestry-informative SNPs yielded were generated successfully in this study from 30 bone samples. Using the ancestry-informative SNP data, the major population bio-ancestry bone samples was determined to be European Ancestry since the bone cases are from Serbia except two samples which shown Asian Ancestry. The targeted sequencing data produces high depths of coverage, thereby enhancing the possibility of successful analysis of challenging forensic specimens such as mixtures and highly degraded DNA samples, since the bones have a range of degradation index (1-40) [11]. Fig 4, Pie chart showing results of the Haplogroup Predictor online tool [2] for the male bone samples Y-STR haplotypes and ancestry SNPs biographic predictor using Illumina Forensic signature Tool. The bone samples is predicted to belong to haplogroup I2a (44%) followed by E1b1b (17%), G2a (11%), R1b (11%), J2a (6%), C2b1a (5%) and N2 (6%), whilst the results for the bones are with a range of probability of (71% -100%), (Table 1 -Supplementary material). Sample number 25 showing results of the Nevgen Haplogroup Predictor for the male bone samples Y-STR haplotypes and ancestry biographic predictor using ForenSeqTM Universal software, it shows almost the same Haplogroup in both tools which is R1B and shows the ancestry Y-Haplogroup with a 32% match in the World Wide in the Y-Haplogroup for this bone sample that is originated from Western European. 5. Conclusion In this work Forensic Signature DNA kit analyses of degraded bone samples indicated that the allele recovery for auSTRs was significantly higher than the GlobalFiler™ kit. All samples sowed concordant results for GlobalFiler™ and ForenSeq™ kit. MPS data showed concordant and superior quality auSTR profiles. The availability of information about the visible phenotypic traits along with identity and ancestry information and traditional STR data can tremendously enhance the value of the MPS analyses in forensic casework. This work has shown clear advantages of MPS analyses of degraded human remains in terms of the amount of relevant information gained for forensic casework and mass disaster victim identification. Overall 230 markers of were detected in all of 30 of the bone samples that includes femur and tooth samples.

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Figure 1. (A & B) Results of 30-bone samples run on FGx-MiSeq showing a cluster density of 866 K/mm2. A total 95% of clusters generated run passed filters, which showed that the reaction was highly successful. The average intensity of the reads was 200000.

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Figure 2. (A & B) DNA Profile of a degraded bone sample (DI = 13) showing a dropout of 7 auSTR loci using GlobalFiler™ kit. MPS results for various markers from the same sample show high efficacy and enhanced discrimination power of Illumina® ForenSeq™ DNA Signature Prep kit

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Figure 3. Phenotype estimation of a Serbian bone sample showing a high prediction for blond hair and blue eyes. The sample was predicted to have an ancestry of Eastern European population correctly

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Figure 4. Pie chart showing NevGen Haplogroup Predictor tool results for 18 male bone samples showing the distribution of

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