Alternate DNA extraction and STR profiling strategies for skeletal and other challenging samples

Abstract

Skeletal remains are often submitted for DNA analysis for human identification (HID) purposes, to either supplement or substitute other forensic identification methods, such as anthropological and odontological analyses. However, the identification of skeletal samples via DNA testing is often challenging and alternate sample processing methods may offer some effective solutions. Current protocols for processing skeletal material involve crushing the bone into a fine powder, which requires specialized equipment, reagents, training, and can pose an increased risk of contamination. Although methods that involve powdering bone tissues for extraction often yield sufficient results for statistical comparisons, bypassing this step can eliminate many of these risks, save time and resources, and also make bone extractions easier for forensic DNA laboratories to implement. Several issues, such as PCR inhibition and DNA degradation, can also make identification more difficult. Therefore, products that provide analysts with more information about sample quality at various stages of the HID process could greatly improve the genotyping process and assist with sample triage and workflow decisions. The main aim of this research was to evaluate the effectiveness of various protocols that eliminate the need to powder bone tissue prior to DNA extraction. A secondary aim of this study was to investigate the benefits of internal STR quality controls for assessing sample quality and determining rework strategies when challenging samples fail to produce complete STR profiles. Several non-powdering DNA extraction methods were tested with human skeletonized remains of varying quality to identify the most efficient protocol to achieve the highest genotyping success. In addition, a broader scope of forensically relevant sample types were genotyped with the Investigator® 24plex QS and GO! kits that include quality controls to test their effectiveness in identifying issues that negatively impact STR success, and guide the most efficient sample rework strategies. As a result of this work, an effective powder-free DNA extraction workflow was identified and shown to be successful with a variety of environmentally challenged skeletal samples. Additionally, we have demonstrated that internal STR quality sensors can simplify STR profile interpretation, help reduce the number of sample reworks, and generate more complete STR profiles when samples are reworked based on the information provided by the quality sensors. These alternate methodological approaches can reduce overall processing time and costs for a wide range of challenging samples subjected to STR typing for human identification purposes. This research has also been used as the scientific basis to amend recommended protocols for some commercial HID products, which will directly benefit the forensic community.