A study of degraded skeletal samples using ForenSeq DNA Signature™ Kit

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. The Illumina® ForenSeq™ DNA Signature MPS Kit, includes 200 genetic loci [2]. The use of NGS 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 Illumina® ForenSeq™ DNA Signature MPS 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

DNA typing from skeletal remains using GlobalFiler™ PCR amplification and Investigator® 24plex QS kits

Since the beginning of our work in 2003 our laboratory has focused exclusively on STR DNA from bone, a powerful tool in missing person cases. In cases such as mass disasters or missing persons, human remains are challenging to identify as they may be fragmented, burnt, recovered from water, degraded, and/or contain inhibitory substances. To address these challenges, this study has evaluated the performance of relatively new STR kits Investigator® 24plex QS kit (Qiagen) and GlobalFiler™ PCR Amplification kit (Thermo Fisher Scientific) by comparing it with current uses of the AmpFLSTR® Identifiler® Plus kit (Applied Biosystems) to obtain genetic information from skeletal remains. We analyzed 20 bone samples of skeletal remains from routine casework submitted for body identifications by law enforcement corresponding using Investigator® 24plex QS kit and GlobalFiler™ PCR Amplification kit, previously analysed AmpFLSTR® Identifiler® Plus kit (Thermo Fisher Scientific). The data indicates that the STR profiles obtained using the GlobalFiler™ and Investigator® 24plex QS kit for analysis of skeletal remains has shown results in an increased number of reportable genetic loci, and provide greater power of discrimination in comparison to the Identifiler® Plus Kit. Advanced extraction and purification techniques, together with more sensitive and robust new amplification kits allowed us to overcome the challenges associated with processing compromised skeletal remains and ultimately obtain full STR DNA profiles in 99% of the bones

Forensic and population genetic analysis of Serbian population using 21 STR loci of GlobalFiler™ PCR amplification kit

Autosomal short tandem repeats (STRs) have been widely used in forensic investigations. Prior to the application of any DNA based identification method, it is essential to estimate the allele frequencies and forensic statistical parameters of targeted STR loci in each population in order to provide a more precise reference database for forensic investigation. The GlobalFiler™ Kit is a multiplex assay that combines the 13 original CODIS loci with 7 non-overlapping loci from the expanded European Standard Set (ESS), as well as the highly discriminating SE33 locus, two Y-based loci and the sex determining maker, Amelogenin. The full complement of loci in the GlobalFiler™ Kit are: D13S317, D7S820, D5S818, CSF1PO, D1S1656, D12S391, D2S441, D10S1248, D18S51, FGA, D21S11, D8S1179, vWA, D16S539, TH01, D3S1358, AMEL, D2S1338, D19S433, DYS391, TPOX, D22S1045, SE33 and a Y-specific insertion/deletion locus (Yindel). The 6-dye GlobalFiler™ PCR Amplification kit (ThermoFisher Scientific) comprises 21 autosomal STRs have already been proven to be able to provide reliable DNA profiling results and enhance the power of discrimination between individuals. In this study, we are presenting an analysis of GlobalFiler STR loci on 209 unrelated individuals from Serbia

DNA analysis from human skeletal remains in forensic casework

To assess our laboratory’s success with skeletal remains and provide a benchmark for the forensic community involved in identification of these remains, we retrospectively examined our ability to develop DNA profiles from the remains analyzed in our laboratory in the last 7 years. Between January 2009 and December 2016, 70 DNA extractions were completed on skeletal remains from routine casework. 92% of skeletal remains analyzed were samples submitted for body identifications by law enforcement and only 8% were samples submitted to answer family identity or historical questions. Overall, the ability to obtain a full or partial profile primarily reflects the difference in the average age and the condition of the samples in these two categories and thus, difference in the quantity and quality of the DNA. We describe here the approximate age and type of remains we have received, whether a full, partial, or no profile was obtained, as well as the condition of the samples

Mutation rate at 13 rapidly mutating Y-STR loci in the population of Serbia

Recently, the interest of the forensic community has been focused on new Y-chromosomal short tandem repeats (Y-STRs), termed Rapidly Mutating Y-STRs (RM-YSTRs), which is able to differentiate between close males belonging to the same paternal lineage due to their high mutation rates. In this study, we have estimated a mutation rate for 13 RM-YSTR in 85 pairs of male relatives in the population of Serbia. We analysed 74 father-son pairs, and 11 twin pairs, to evaluate the capacity of distinguishing between male subjects within a single lineage. Each father-son couple was previously confirmed by autosomal STRs testing (AmpFℓSTR® Identifiler Plus™ kit, Applied Biosystems) with paternity probability ≥99.99% and also confirmed monozygotic or dizygotic twins. Results showed that, in the 74 father-son pairs 23 mutations were detected of which 22 were one-step mutations and 1 was two-step mutation, while in the 11 twin pairs 1 mutation was observed in one dizygotic twin pair. Five father-son pairs were found to have mutations at two loci, while one pair at four loci. Overall, the most mutable markers were DYF399S1, DYF387S1, DYF403S1a and DYS612. Our findings are encouraging and concur with previous studies showing that by RM-YSTR typing the discrimination power of male relatives could be considerably increased in comparison to every YSTR markers commonly used in forensic genetics

Genetic characterization of 27 Y-STR loci with the Yfiler ® Plus kit in the population of Serbia

Letter to the Edito

Rapidly mutating Y-STRs population data in the population of Serbia and haplotype probability assessment for forensic purposes

Recent research with Rapidly Mutating Y-STRs (RM Y-STRs) have shown that these loci provide substantially higher haplotype diversity and haplotype discrimination capacity in worldwide populations when compared with the Y-STRs commonly used in genetic forensics. The aim of this study was to develop an allelic frequency database for the population of Serbia in order to evaluate the resolution power of 13 RM Y-STRs. A total of 279 unrelated males from the population of Serbia were typed with 13 RM Y-STRs loci: DYF387S1, DYF399S1, DYF403S1a/b, DYF404S1, DYS449, DYS518, DYS526a/b, DYS547, DYS570, DYS576, DYS612, DYS626 and DYS627. A high Y-STR haplotype diversity was found (0.999768) in our samples. As expected, the RM Y-STR loci showed high genetics diversity (GD) values (>0.73) in the Serbian population. The highest GD was observed for the locus DYF399S1 (0.991), followed by loci DYF403S1a (0.976), DYF387S1 (0.907) and DYF404S1 (0.899). Based on the results of this study, the RM Y-STR loci showed remarkable haplotype resolution power in the population of Serbia, high genetics diversity and, therefore, demonstrating their usefulness in forensic identification cases