Analysis of paterniy cases with a single exclusion in a genetic marker using precision ID GLOBALFILER™ NGS STR PANEL v2

Paternity test results can sometimes evidence incompatibilities in the allelic transmission from parents to children, such as the presence of a single exclusion in one specific genetic marker, revealing a mismatch between the genetic profiles of the biological parent and the offspring. In these cases, it is important to determine whether the exclusion could be the result of a mutation or other factors as null or silent alleles. Capillary electrophoresis (CE) is the traditional method used in forensic genetics to analyze STRs (Short Tandem Repeats), however it is not possible to know the exact allele number variation due to the lack of sequence data. The application of Next-Generation Sequencing (NGS) technology may provide additional information, since it allows to detect and sequence simultaneously SNPs (Single Nucleotide Polymorphisms) present in the flanking regions and also distinguish isometric alleles with the same length but different sequences, that were misinterpreted as homozygous. In this study, a set of reference samples (buccal swabs and blood stains), previously amplified with the GlobalFiler™ PCR Amplification Kit and sequenced by CE on the 3500 Genetic Analyzer, were selected from paternity cases with a single exclusion, reported after GeneMapper ID-X Software analysis. All samples were automatically prepared with the Precision ID GlobalFiler™ NGS STR Panel v2 on the Ion Chef™ System, followed by sequencing on the Ion S5™ System and finally Converge™ Software analysis, according to the manufacturer’s instructions. The aim was to verify if the NGS methodology provides valuable information in these paternity cases and to identify the parental origin of a mutant allele. The NGS results were in concordance with those obtained by CE. In addition, this methodology demonstrated to be useful to clarify the paternity cases, because it enables a higher power of discrimination through 9 additional multi-allelic STRs, in a total of 35 markers instead of 24 markers of the GlobalFiler™ PCR Amplification Kit used in the traditional method. Therefore, the Precision ID GlobalFiler™ NGS STR Panel v2 shows to be a powerful method for kinship analyses and typing reference samples.