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Removing PCR Artifacts in Forensic DNA Analysis

Event Dates
Event Duration
1 hour

​The use of short-tandem repeats (STR) in forensic DNA analysis has been successful in most situations. However, this technology has several important complicating factors that are intrinsic to current STR genotyping methods, such as spurious background peaks resulting from PCR stutter, co-migration, signal oversaturation, and machine noise. Of particular concern are PCR stuttering artifacts, which arise from slippage of the DNA polymerase of the DNA template. Damaged or degraded DNA is particularly prone to this form of error due to the prevalence of DNA adducts that cause erroneous base pairings and enzyme stalling. Together, these factors dramatically increase the difficulty to identify individuals occurring at a minor allele frequency (MAF) of less than 10%, mixtures with >2 people.

The advent of massively parallel sequencing (MPS) has promised to revolutionize forensic DNA analysis. Unlike conventional STR analysis, which simply reports the average genotype of an aggregate population of molecules, MPS technology digitally tabulates the sequence of many individual DNA fragments, thus offering the unique ability to detect MAFs within a heterogeneous DNA mixture. Unfortunately, most MPS protocols rely on PCR during sample preparation, which, in conjunction with damaged DNA samples, increases the rate of stutter and base misincorporations and limits its utility for effective forensic analysis. To overcome the high error rate resulting from PCR and facilitate the genotyping of damaged DNA samples and heterogeneous DNA mixtures, we recently developed a highly accurate MPS 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 methods. The DS method also specifically removes the confounding effects of stutter artifacts through a unique solution that can be incorporated into any standard MPS workflow.

This seminar will review the basics of MPS technologies and describe Duplex Sequencing and its current uses, as well as applications for highly accurate forensic genotyping.

Date Created: August 24, 2016