Webinar: Development of a Probe Capture NGS System for Forensics

Registration is open for a free, two-hour webinar that will explain the development of a probe capture NGS system for analyzing complex and low-level mixtures, highly degraded, and limited DNA samples.

Forensic biological samples encountered in mass disasters, missing persons, and forensic cases can often be highly compromised (degraded, limited, or mixed). Alternative markers such as nuclear Single Nucleotide Polymorphisms (SNPs) or mitochondrial DNA (mtDNA) can be analyzed for increased genotyping success in cases where STR analysis using conventional electrophoretic methods fail. Nuclear SNPs serve as suitable targets for analysis of degraded DNA since the targeted variation is a single base pair. However, in some cases nuclear DNA may be too limited or degraded for SNP analysis using PCR based methods.

Analyzing mitochondrial DNA is advantageous for degraded and limited samples (such as telogen hairs) since the mitochondrial genome occurs in high copy number per cell. Current approaches include sequencing the hypervariable regions (HVI/HVII) by Sanger sequencing. The maternal inheritance pattern of mtDNA and sequence information from only the HV regions provides limited discrimination power, particularly in the Caucasian population. Sanger sequencing is also limiting in that it often fails to detect low level heteroplasmy as well as mixtures which are common in forensic samples. New methods using next-generation sequencing (NGS) technologies can be used to overcome some of these limitations.

To analyze challenging forensic DNA samples, we have developed probe capture NGS systems for targeting the entire mitochondrial genome and over 450 nuclear polymorphisms. This approach uses DNA probes to enrich targeted regions from randomly fragmented DNA libraries for clonal, massively parallel sequencing, thereby maximizing recovery of short DNA fragments characteristic of forensic samples. The clonal sequencing aspect of NGS allows for analysis of the components of a mixture separately and by counting the number of sequence reads assigned to each individual contributor of the mixture. We have successfully applied this system to sequence the entire mitochondrial genome of limited and highly degraded DNA from hair and bones as well as mixtures. 100% sequence coverage of the mitochondrial genome with a > 95% on-target rate was achieved for blood derived DNA samples and controls varying in DNA amounts ranging from 10pg-1ng. The mitochondrial probe capture NGS assay was also successfully applied to the analysis of highly degraded DNA from telogen hairs and bone specimens. We have also analyzed contrived mixtures at varying ratios using the mtDNA probe capture NGS system and can detect and resolve minor components of a mixtures as low as 5-10% depending on the read depth. We have also demonstrated the feasibility of two different software programs for reconstructing the mitochondrial genome sequence of the contributors in a mixture. In collaboration with Dr. Richard Green at UC Santa Cruz a software program (mixemt) was developed to aid in the analysis of the mtDNA mixtures. This software tool uses an expectation-maximization algorithm to resolve major and minor haplotypes in a mixture.

This event is hosted by Dr. Cassandra D. Calloway, an assistant scientist at Children's Hospital Oakland Research Institute, and assistant professor in the Department of Pediatrics in the School of Medicine at the University of California, San Francisco.

Click here to learn more and register now.

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