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                            Funding & Awards
 Validation of a Probe Capture Next-Generation Sequencing Assay for Whole Mitochondrial Genome Analysis
Award Information
Awardee
              Children''s Hospital & Research Center at Oakland
Award #
              2015-DN-BX-K053
Funding Category
              Competitive
Location
                    Oakland, CA
Awardee County
              Alameda
Congressional District
              13
Status
              Closed
Funding First Awarded
              2015
Total funding (to date)
              $758,184
Original Solicitation
              NIJ FY 15 Research and Development in Forensic Science for Criminal Justice Purposes
Description of original award (Fiscal Year 2015, $758,184)
As submitted by the proposer: 

Many forensics samples encountered in criminal cases or in the identification of missing persons cannot be successfully analyzed with nuclear (chromosomal) genetic markers and would benefit greatly from analysis of mitochondrial (mt) DNA. Many of the properties of mtDNA make it very valuable for the analysis of forensic specimens with limited and/or degraded DNA, for the deconvolution of forensic mixtures, and for identification of missing persons. These properties include high concentration (thousands of copies per cell), haploid nature (one sequence per contributor in mixture), and matrilineal inheritance (maternal relatives as reference for missing persons). One reason that mtDNA has not been used more widely in forensic analyses is the perception that it is not very discriminating/informative. This perception is based, in part, on the fact that most current mtDNA forensics analyses rely solely on polymorphisms in the HVI/HVII regions and use the Sanger sequencing method. The massively parallel and clonal nature of Next Generation Sequencing (NGS) makes the analysis of the whole mitochondrial genome feasible as well as the deconvolution of mixed samples. We have successfully developed a solution phase probe capture and NGS method on the Illumina MiSeq for targeted enrichment and deep sequencing of the entire mitochondrial genome for increased discrimination power. Using this probe capture NGS assay, 100% sequence coverage of the mitochondrial genome with an ~95% on target rate was achieved. The input DNA amount was successfully lowered to the forensically relevant level of 10 pg demonstrating the high sensitivity of the method. Moreover, a DNA fragmentation method using mechanical shearing (Covaris) was optimized and shown to be DNA quantity and quality independent, essential for preparation of highly degraded or limited samples. This optimized fragmentation method coupled with the probe capture enrichment assay can be used for analysis of degraded samples often encountered in forensic cases as well as mixtures with increased sensitivity and discrimination potential. However, as with any new technology/method, a developmental validation must first be completed prior to forensic use.

We propose to complete a developmental validation and coordinate testing/internal validation studies at two external sites (a government and a commercial laboratory). We will also validate custom commercial software and develop interpretation guidelines for whole mitochondrial genome NGS analysis. Successful completion of the required developmental validation studies proposed here will allow for implementation of this robust, highly informative system for analyzing mtDNA polymorphism in any forensic laboratory following internal validation.

This project contains a research and/or development component, as defined in applicable law.

ca/ncf



  
Grant-Funded Publications
        
                      Validation of a Probe Capture Next-Generation Sequencing Assay for Whole Mitochondrial Genome Analysis
                  Target Capture Enrichment of Nuclear SNP Markers for Massively Parallel Sequencing of Degraded and Mixed samples
                  Applications of Probe Capture Enrichment Next Generation Sequencing for Whole Mitochondrial Genome and 426 Nuclear SNPs for Forensically Challenging Samples

  
Date Created: September 17, 2015
    
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