Massively Parallel Sequencing Platform for High Accuracy Variant Detection
Abstract
The use of short-tandem repeats (STR) in forensic DNA analysis has been successful in most situations. However, this technology has several important limitations, including difficulty in identifying individuals occurring at a minor allele frequency (MAF) of less than 10%, mixtures with >2 people, and damaged DNA samples. The advent of Next-Generation Sequencing (NGS) has promised to revolutionize forensic DNA analysis due to increased sensitivity in detecting lower MAFs. Unfortunately, NGS relies on PCR during sample preparation, which, in conjunction with damaged DNA samples, introduces a significant amount of artifacts, limiting its utility for forensic analysis. To overcome the high error rate of NGS and facilitate the genotyping of damaged DNA samples and heterogeneous DNA mixtures, we recently developed a highly accurate NGS methodology, termed Duplex Sequencing (DS). DS dramatically improves the ability to genotype difficult forensic DNA samples and delivering an unprecedented >10,000-fold improvement in accuracy compared to conventional NGS. DS exploits the inherent complementarity of double-stranded DNA. Unique molecular tags are used to label each fragmented DNA molecule with complementary, double-stranded nucleotide sequences. This allows every sequence to be traced back to one of the two strands of a unique double-stranded DNA molecule. The two complementary sequences derived from the two strands of each unique DNA duplex are then compared. Apparent mutations introduced during PCR or arising from DNA damage occur only one of the two DNA strands and recognized and eliminated as artifactual. The proposed project will develop and adapt the DS technology for use in a forensic laboratory environment specializing in forensic DNA analysis.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Oct 22, 2018
- Source ID
- W911NF1710158
Entities
People
- Scott Kennedy
Organizations
- Army Contracting Command
- United States Army
- University of Washington