Extraction efficiency testing of degraded bone samples: Comparing four DNA extraction methods for downstream massively parallel sequencing applications

WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
Emily Lucia Deem (Creator)
Western Carolina University (WCU )
Web Site: http://library.wcu.edu/
Frankie West

Abstract: In recent years, investigative genetic genealogy (IGG), which involves the use of genealogical methods combined with DNA analysis to make potential familial matches, has become an important tool in solving cold and active cases. These cases can involve the identification of a perpetrator or the identification of missing persons. Estimates show that approximately 4,400 unidentified bodies are recovered each year, and up to one quarter of those individuals remain unidentified after one year. Traditionally, forensic DNA amplification methods have relied on the need to amplify 100-450 base-pair targets, specifically, short tandem repeats (STRs), for forensic profiles. With genetic genealogical approaches, smaller targets, such as single nucleotide polymorphisms (SNPs), have shown potential as tools for identification. Traditional extraction protocols for forensic DNA have focused on maximizing DNA recovery with the intent of amplifying larger STR targets. On the other hand, ancient DNA techniques have focused efforts on recovering smaller DNA fragments, like SNPs, and indeed have shown recovery of even highly degraded samples in excess of 400,000 years. This study aims to compare the extraction success and efficiency of one ancient DNA (aDNA) extraction technique from Rohland et al. (2018) and three forensic DNA extraction techniques, PrepFiler® BTA Forensic DNA Extraction Kit from Applied Biosystems, the Bone DNA Extraction Kit, Custom from Promega, and the InnoXtract™ from InnoGenomics, on compromised bone samples for the purposes of massively parallel sequencing (MPS). Quantitative PCR was used to compare the extraction performance of the protocols, while an MPS-based assay, the Ion AmpliSeq™ PhenoTrivium Panel, was used to assess informative characteristics, such as phenotype and biogeographic ancestry, for an investigation. The Rohland and modified PrepFiler protocols showed the most success in terms of DNA recovery and sequencing. These results show the utility of an ancient DNA extraction method in MPS research and the success of a widely used forensic method. The results of this study may add to the process of determining the most appropriate extraction method for massively parallel sequencing applications such as IGG in forensic contexts.

Additional Information

Language: English
Date: 2022
degraded samples, DNA extraction, human identification, massively parallel sequencing, SNPs
Forensic pathology
Forensic genetics
Single nucleotide polymorphisms
Nucleotide sequence

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