Preparing for shotgun sequencing in forensic genetics – Evaluation of DNA extraction and library building methods

Shotgun sequencing can be a powerful tool in forensic genetics, enabling comprehensive genetic analyses of biological samples for human identification (HID), forensic DNA phenotyping, ancestry inference, and forensic investigative genetic genealogy (FIGG). This study evaluated the performance of shotgun sequencing of typical forensic reference samples (whole blood or punches from FTA cards) extracted with four commonly used DNA extraction methods. The four DNA extraction methods were paired with three different library building methods to determine the best combination of procedures and their impact on the quality and quantity of the sequencing reads. Shotgun sequencing was performed on an Illumina NovaSeq 6000 system. The data was analysed for coverage, total number of reads, mapped reads, median insert size, and presence of forensically relevant loci, including short tandem repeats (STRs), ancestry informative markers (AIMs), single nucleotide polymorphisms (SNPs) associated with pigmentary traits (HIrisPlex-S), SNPs on the Y chromosome, and SNPs used for FIGG. The highest quality of sequencing data was achieved using the combination of EZ1&2 DNA Investigator Kit extractions and a double-stranded library building method, or the combination of Chelex® or PrepFiler Express™ Forensic DNA Extractions with a single-stranded library building protocol. The combination of EZ1&2 DNA extraction and double-stranded library building yielded the largest number of genotypes. As many as 36 STRs, 162 AIMs, 41 HIrisPlex-S SNPs, 85,712 Y-SNPs, and 1.3 million FIGG SNPs were genotyped in one experiment. On the contrary, the combination of Chelex® or PrepFiler™ together with a double-stranded library building method generated relatively few genotypes and low-quality results. The single-stranded library building protocol could be applied to EZ1&2 DNA Investigator Kit extractions of DNA on FTA cards but was inefficient and generated low-accuracy data when the sample material was whole blood. In conclusion, this study highlights the importance of combining the different forensic DNA extraction methods with appropriate shotgun sequencing library preparation approaches to optimise both the quantity and quality of forensically relevant DNA data.