Recent advances in massively parallel sequencing (MPS), provide advantages in profiling DNA from skeletal human remains, particularly old ones due to enhanced sensitivity and simultaneously analysis of much greater number of loci then the current capillary electrophoresis based technology. The ForenSeq™ DNA Signature kit, includes autosomal STRs, Y STRs, X STRs, Identity SNPs and Ancestry SNPs [[1]Next generation sequencing and its applications in forensic genetics.]. 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]Chaitanya L. Pajnič I.Z. Walsh S. Balažic J. Zupanc T. Kayser M. Bringing colour back after 70 years: Predicting eye and hair colour from skeletal remains of World War II victims using the HIrisPlex system.

Forensic Sci. Int. Genet. 2017; 26: 48-57

,[9]Grunenwald A. Keyser C. Sautereau A.-M. Crubézy E. Ludes B. Drouet C. Adsorption of DNA on biomimetic apatites: toward the understanding of the role of bone and tooth mineral on the preservation of ancient DNA.

Appl. Surf. Sci. 2014; 292: 867-875

]. The greatest challenge faced in the analysis of degraded samples, is the potential for any extracted DNA to have come from a source other than the target [[7]Applications of next generation sequencing in molecular ecology of non-model organisms.

Heredity. 2011; 107: 1-15

,[12]Meyer M. Fu Q. Aximu-Petri A. Glocke I. Nickel B. Arsuaga J.-L. Martínez I. Gracia A. De Castro J.M.B. Carbonell E. A mitochondrial genome sequence of a hominin from Sima de los Huesos.

Nature. 2014; 505: 403-406

]. The larger loci (>200bp) are therefore the first to be affected, and are commonly lost [[1]Next generation sequencing and its applications in forensic genetics.,[11]King T.E. Fortes G.G. Balaresque P. Thomas M.G. Balding D. Delser P.M. Neumann R. Parson W. Knapp M. Walsh S. Identification of the remains of King Richard III.

Nat. Commun. 2014; : 5

,[12]Meyer M. Fu Q. Aximu-Petri A. Glocke I. Nickel B. Arsuaga J.-L. Martínez I. Gracia A. De Castro J.M.B. Carbonell E. A mitochondrial genome sequence of a hominin from Sima de los Huesos.

Nature. 2014; 505: 403-406

]. Recent advances in (MPS), provides advantages in the analysis of DNA from human remains [[7]Applications of next generation sequencing in molecular ecology of non-model organisms.

Heredity. 2011; 107: 1-15

,[14]Rockenbauer E. Hansen S. Mikkelsen M. Børsting C. Morling N. Characterization of mutations and sequence variants in the D21S11 locus by next generation sequencing.

Forensic Sci. Int. Genet. 2014; 8: 68-72

]. 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 [[3]Chaitanya L. Pajnič I.Z. Walsh S. Balažic J. Zupanc T. Kayser M. Bringing colour back after 70 years: Predicting eye and hair colour from skeletal remains of World War II victims using the HIrisPlex system.

Forensic Sci. Int. Genet. 2017; 26: 48-57

]. 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 [[13]Morozova O. Hirst M. Marra M.A. Applications of new sequencing technologies for transcriptome analysis.

Annu. Rev. Genomics Hum. Genet. 2009; 10: 135-151

,[14]Rockenbauer E. Hansen S. Mikkelsen M. Børsting C. Morling N. Characterization of mutations and sequence variants in the D21S11 locus by next generation sequencing.

Forensic Sci. Int. Genet. 2014; 8: 68-72

,[20]Zeng X. King J.L. Stoljarova M. Warshauer D.H. Larue B.L. Sajantila A. Patel J. Storts D.R. Budowle B. High sensitivity multiplex short tandem repeat loci analyses with massively parallel sequencing.

Forensic Sci. Int. Genet. 2015; 16: 38-47

]. In the current work presented here, we have investigated the performance of the ForenSeq™ DNA Signature kit, on a set of degraded skeletal DNA samples from Serbia.