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New Methods for Next-Generation Sequencing Analysis of Damaged and Mixed DNA Samples

Identifying individuals through next generation DNA sequencing may involve degraded or mixed samples. A researcher has developed a technique to make fragmented DNA more readable, and a method to help pull individual profiles from mixed samples.
Date Published
April 6, 2020

A person’s genome consists of 46 long DNA molecules in the nuclei of their cells, as well as a long loop of DNA found in the mitochondria. Molecules of nuclear DNA are called chromosomes and they come in 23 matching pairs, one of each pair from each parent. Mitochondrial DNA (mtDNA) is inherited from the mother and is hundreds of times more plentiful than nuclear DNA in most cells.

When a human genome is exposed to the elements, such as water, ultraviolet radiation, or high temperatures, the DNA molecules undergo changes that make them unreadable. The molecules break into smaller pieces and some of those breaks cut across key identifying regions (markers), such as the short tandem repeats (STRs) used in forensic analysis. They also develop new molecular bonds that prevent important enzymes from functioning when the molecules are sequenced in a laboratory.

Over time, increasing numbers of DNA molecules in a sample lose their utility, and the longer or rarer the marker, the more likely it will not be sequenced during laboratory analysis. Unfortunately this includes STRs, but scientists have found two alternative approaches that can enable human identification. The first approach is to use single nucleotide polymorphisms (SNPs, called “snips”) since, as their name indicates, the identifying element is a single unit of the DNA molecule, not the dozens that are needed for an STR. The second approach is to focus on mtDNA, since it is so much more plentiful than nuclear DNA.

A problem with using mtDNA for human identification is that it is inherited as a whole from the mother, and it undergoes no mixing with other versions of mtDNA in the population. As a result, there are far fewer unique versions of mtDNA, and its identifying markers are less conclusive in human identification.

To overcome the difficulties of sequencing fragmented and mixed DNA, Cassandra Calloway, a scientist at Children’s Hospital Oakland Research Institute (California), developed a novel approach that focused on SNPs in the nuclear DNA. She used a technique to gather and concentrate the SNP-containing DNA fragments before sequencing. This concentrating step is important, as the many small fragments of damaged DNA lower the efficiency and precision of sequencing processes. This technique, called “probe capture,” uses short, laboratory-manufactured DNA or RNA sequences that match the DNA sequences next to SNPs of interest, as well as a protein-vitamin complex and tiny magnetic beads. The manufactured sequences bond to the DNA fragments in the sample that have the SNPs of interest, and the protein-vitamin complex attaches these fragments to the beads. Finally, a magnet pulls the beads and fragments to the side of the tube and holds them there while everything else is washed away.

Calloway and her team found that not only was this technique highly successful at allowing them to sequence hundreds of different SNPs from highly degraded samples, but in two-person mixtures the ratio of the original mixture was discernible in the ratio of captured and sequenced SNPs 85­­ to 100 percent of the time, thus allowing them to provide a reliable SNP profile for each person. 

In a second study funded under this award, Calloway and her colleagues used a new analytic method to generate individual DNA profiles from multiperson mixtures of highly fragmented mtDNA. Normally, mtDNA does not pose a problem in mixtures, as each person has only one version of mtDNA and it consists of a single molecule. However, in samples with multiple donors (such as a mixed blood stain from a victim and suspect), or where the mtDNA has fragmented due to age or other damage, it is impossible to definitely associate unique, identifying mutations from one region of the mtDNA with those from other regions for each contributor. Calloway developed an analytic tool that creates accurate profiles from a variety of mixture proportions by using two pieces of information: (1) the proportion that each mutation is found in the next-generation sequencing reads and (2) population and ancestry information on the most likely combinations of mtDNA mutations. Either of these novel methods would allow for DNA profiling of samples too aged or damaged, such as those from the 9/11 attacks or the recent California wildfires, to produce any identifying information using standard forensic STRs.

About This Article

The research described in this article was funded by NIJ grant 2013-MU-MU-K044 awarded to Children’s Hospital Oakland Research Institute. This article is based on the grantee report “Development of Probe Capture Next-Generation Sequencing Assays for Degraded DNA” by Cassandra D. Calloway, principal investigator, Children’s Hospital Oakland Research Institute.

Date Published: April 6, 2020