U.S. flag

An official website of the United States government, Department of Justice.

Dot gov

The .gov means it’s official.
Federal government websites always use a .gov or .mil domain. Before sharing sensitive information online, make sure you’re on a .gov or .mil site by inspecting your browser’s address (or “location”) bar.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Validation of a Probe Capture Next-Generation Sequencing Assay for Whole Mitochondrial Genome Analysis

Award Information

Award #
2015-DN-BX-K053
Location
Awardee County
Alameda
Congressional District
Status
Closed
Funding First Awarded
2015
Total funding (to date)
$758,184

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

Date Created: September 17, 2015