Preparing for Shotgun Sequencing in Forensic Genetics: Optimizing DNA Extraction and Library Preparation
Organized by: International Forensic Scientist Awards
Website: forensicscientist.org
13th Edition of Forensic Scientist Awards 28-29 August 2025 | Berlin Germany
Shotgun sequencing has emerged as one of the most powerful tools in forensic genetics, offering a comprehensive approach to analyzing biological samples for a wide range of investigative purposes. From human identification (HID) and forensic DNA phenotyping to ancestry inference and forensic investigative genetic genealogy (FIGG), shotgun sequencing can deliver massive amounts of genetic information in a single run—if the right preparation steps are taken.
This study set out to evaluate how different DNA extraction methods and library building protocols influence the quality and quantity of data obtained through shotgun sequencing in a forensic context. Researchers worked with common forensic reference samples—either whole blood or FTA card punches—and applied four widely used DNA extraction techniques. Each extraction method was then paired with three different library preparation approaches to identify the most effective combinations for producing high-quality sequencing reads.
Shotgun sequencing was performed on the Illumina NovaSeq 6000 platform, and the resulting data were analyzed for:
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Coverage
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Total number of sequencing reads
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Mapped reads
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Median insert size
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Recovery of forensically relevant loci
The analysis focused on genetic markers essential to forensic casework, including short tandem repeats (STRs), ancestry informative markers (AIMs), SNPs linked to pigmentation traits (via HIrisPlex-S), Y-chromosome SNPs, and SNPs used for FIGG applications.
Key Findings
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The EZ1&2 DNA Investigator Kit paired with a double-stranded library building protocol produced the highest quality sequencing data overall, yielding the largest number of genotypes.
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The Chelex® and PrepFiler Express™ Forensic DNA Extraction methods also performed well when paired with single-stranded library building protocols, producing data of comparable quality in certain conditions.
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In the most successful experiment, the EZ1&2 and double-stranded combination enabled the recovery of:
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36 STRs
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162 AIMs
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41 HIrisPlex-S SNPs
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85,712 Y-SNPs
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1.3 million FIGG SNPs
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Conversely, pairing Chelex® or PrepFiler™ with a double-stranded protocol resulted in low-quality data and fewer genotypes.
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Single-stranded protocols could be applied to EZ1&2 extractions from FTA cards with moderate success, but proved inefficient for whole blood samples.
Why It Matters for Forensic Science
These results highlight a critical takeaway for forensic laboratories: the combination of DNA extraction method and library preparation strategy can significantly impact sequencing outcomes. Simply put, even the most advanced sequencing technology will not deliver its full potential without careful optimization of upstream processes.
For casework applications—where sample quality, quantity, and preservation vary widely—this study provides a clear evidence-based guide for selecting methods that maximize both data yield and accuracy. The right pairing not only improves recovery of STRs and SNPs but also enhances the ability to perform complex analyses like FIGG and phenotype prediction from challenging samples.
As forensic science continues to integrate high-throughput sequencing into its workflows, understanding these preparatory variables will be key to unlocking its full investigative power.
Conclusion
This study demonstrates that the success of shotgun sequencing in forensic genetics depends heavily on the synergy between DNA extraction and library preparation methods. Among the tested combinations, the EZ1&2 DNA Investigator Kit with a double-stranded library building protocol consistently delivered the highest quantity and quality of forensically relevant genetic data, making it the most effective choice for comprehensive human identification and investigative applications. While certain single-stranded protocols paired with specific extraction methods, such as Chelex® or PrepFiler Express™, can yield good results under particular conditions, mismatched combinations can severely compromise sequencing performance.
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