Forensic DNA genotyping by means of next generation sequencing. Analysis of Autosomal STRs of a Norwegian population sample using the ForenSeq FGx system

Abstract

Population databases containing allele frequencies of the genetic markers used for DNA-profiling are necessary for forensic geneticist to be able to perform statistical calculations on the statistical weight of DNA-evidence. However, allele frequencies differ from population to population, it is therefore important to establish population databases for specific graphical areas or population groups. The Center of Forensic Genetics is currently using a method based on PCR and capillary electrophoresis for DNA-profiling, but wants to establish a method based on deep sequencing. The purpose of this study was therefore to establish a population database for a Norwegian population with allele frequencies of autosomal STR markers used for DNA-profiling with the new NGS-based method. Samples from a previously established biobank were used to obtain DNA profiles for all 231 forensic genetic markers included in the ForenSeq DNA Signature Prep kit. Validation data of the ForenSeq FGx system was also assessed, focusing only on the 27 autosomal STR included in the kit. The Norwegian population database for autosomal STRs was established, with frequencies of both length-based and sequence-based allele variants. There is an increase in the number of sequence-based allele variants compared to length-based allele variants for many markers, meaning the power of discrimination can be raised when using the same number of markers when using a deep sequencing method. A reproducibility and sensitivity study of the ForenSeq FGx system was also conducted. They showed that the system produces 100% reproducible genotypes when sequencing the same samples more than once. The sensitivity study showed that with the ForenSeq FGx system a complete DNA-profile can be obtained for 125 pg DNA, and that approximately 50 % of alleles can still be called with as little as 15.625 pg DNA. To check if the two methods, Signature Prep and the NGM Select, could produce the same autosomal genotype results, a concordance study was performed. Almost full concordance was found between the two methods, only three alleles had discordances. The discordance is probably because of two different primers being used for the kits. They might been bound differently an therefor called different autosomal genotypes.