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NGS has the potential to be a promising technology for recovering genetic information from challenging\ud specimens in forensic genetics. In order to understand the role of DNA damage on the outcome of NGS, we\ud investigated the performance of ForenSeqTM DNA Signature kit, Illumina (in its pre-commercial version)\ud on a set of in vitro degraded trial DNA samples. After DNA quantification by qPCR, duplicate analyses of\ud the samples were carried out. The resulting molecular products were then sequenced by using MiSeq1\ud system (Illumina) and analyzed using ForenSeqTM Universal Analysis Software (Illumina). The coverage\ud and error rate of the NGS data obtained from the degraded samples were compared to the ones gathered\ud from the unmodified DNA. The NGS data showed that the ability of recovering genotypes and the\ud frequency of analytical artifacts are strongly influenced by the degree of damage of the template. NGS was\ud able to call 46–17% of the STR loci and 68–26% of the SNPs in the degraded samples. In addition, when the\ud genotypes from the degraded samples were compared to the ones recovered from the unmodified control\ud DNA, correct typing was achieved from 39 to 4% of the STRs and from 55 to 13% of the SNPs.\ud These data show that NGS is a powerful method for gathering genetic data from samples which failed\ud the conventional approaches, even if in this experiment the risk of mistyping seems not to be negligible\ud (up to 2%)

In a standard paternity testing, mother, child, and alleged father are analyzed with STR markers using commercially available kits. Since Italian civil legislation does not have thresholds to confirm a paternity, paternity is practically proven when likelihood ratio increases prior probability of paternity to posterior, accepted by court as sufficient. However, in some cases the number of markers included in a commercial kit may be insufficient to conclusively prove or disprove a relationship between individuals, especially when complex family scenarios are suspected or indirect analyses are required. Additional genetic information can increase the values of the likelihood ratio regarding the detection of true parental relationships in a pedigree, while reducing the chances of false attributions (e.g. false paternities). In these cases the introduction of a 26Plex amplification system allows to examine 23-26 additional markers depending on the commercial kit used, thus increasing the statistical power of the kinship analysis. The PCR conditions were optimized for a multiplex amplification system and a new generation CE instrument. In order to demonstrate the utility of additional STRs markers, four complex kinship cases are presented.

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