Development of Probe Capture Next-Generation Sequencing Assays for Degraded DNA

As submitted by the applicant: Samples with degraded or limited DNA are often encountered in mass disaster, missing person, and forensic cases and can present major interpretation challenges for standard STR markers. The most common approach taken to improve genotyping success of these samples is to use mini STR primer sets. However, mini STRs have limited success for analysis of highly degraded DNA from postmortem tissue and bones exposed to extreme or harsh conditions or limited and degraded DNA from shed hairs. Alternative markers such as nuclear SNPs or mitochondrial DNA (mtDNA) can be used for increased genotyping success. Highly multiplexed nuclear SNP panels have been developed for forensic applications and have been implemented with some success to supplement STR data in several recent mass disaster and mass fatality cases including the identification efforts for the world trade center victims. However, in some cases where DNA is highly degraded or limited, nuclear DNA analysis fails. Mitochondrial DNA testing is most useful in these cases because of its 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. Our preliminary studies show that Next Generation Sequencing (NGS) coupled with a probe capture system can be used for deep sequencing the entire mitochondrial genome. This probe capture technique has the potential to be successfully applied to both nuclear and mtDNA analysis of degraded samples since capture efficiency is not dependent on specific priming sites like PCR based methods. Based on our preliminary results, we propose to 1) develop and optimize a probe capture enrichment assay for deep sequencing the entire mitochondrial genome and nuclear SNP markers and 2) analyze highly degraded and limited forensically relevant samples including shed hairs. Our specific objectives are to 1) optimize a fragmentation method using mechanical shearing for both degraded and reference samples, 2) optimize a rapid DNA library preparation method for both 454 and Illumina NGS sequencing, 3) develop and optimize a probe capture assay for enrichment and deep sequencing of the whole mitochondrial genome using 454 and Illumina NGS technologies, 4) show proof-of-principle of analysis of telogen hairs using probe capture enrichment for nuclear SNPs and 5) develop custom software for analyzing NGS data for forensic applications and markers. Upon successful completion of the project, the probe capture mitochondrial genome enrichment and NGS assays will be evaluated by a forensic laboratory for possible implementation. These assays have the potential to greatly improve the discrimination power compared to current mitochondrial HVI/HVII sequence analysis as well as to increase the number of degraded samples successfully genotyped. If the proof-of-principle using probe capture targeting nuclear SNPs to analyze degraded and limited DNA from a single shed hair is successful, the application to routine hair analysis would greatly advance the forensic field. The NGS software developed for this project will be customized for forensic applications and will be made publicly available. The mtDNA and nuclear SNP probe capture enrichment and NGS assays can be used to supplement STR data or as standalone assays. The proposed capture enrichment coupled with NGS is an ideal system for analysis of degraded and has broad applications, including missing persons, mass disasters, forensic hair analysis, and low template DNA.

ca/ncf