In circumstances where potential crime scene evidence such as hair or bone might be old or degraded, forensic scientists rely on DNA from a cell’s mitochondria—an organelle that has its own genome separate from the “human genome” in the cell’s nucleus. Now, the National Institute of Justice has awarded a team of researchers from Penn State $770,000 to sequence the mitochondrial genomes of 10,000 Pennsylvanians. This will more than triple the size of the existing database and provide a crucial point of reference for use in human identification cases.
“When we talk about DNA, we typically think of DNA from the nucleus of a cell, which is unique from person to person,” said Mitchell Holland, professor of biochemistry and molecular biology and of forensic science at Penn State and leader of the research team. “This type of DNA can be extracted from the root of a hair, but most hair found at crime scenes does not contain the root, and the nuclear DNA within hair shafts is usually degraded. Mitochondrial DNA doesn’t have issues with degradation within the hair shaft, so it provides different but useful information.”
Nuclear DNA is inherited from both parents and intermixes in unique combinations, so, according to Holland, the likelihood of finding another person with the same DNA at a specific set of points along the genome—called a DNA profile—can be one in a septillion, more than seven factors of magnitude rarer than one in a million. By contrast, mitochondrial DNA is inherited solely from the mother, so a person’s mitochondrial genome is typically identical to that of their mother, siblings and others in that maternal line.
“With mitochondrial DNA, the likelihood of finding a person with the same DNA profile is usually one in tens of thousands, or maybe one in hundreds of thousands,” said Erin Brownfield, graduate student at Penn State. “So, matching a person’s mitochondrial DNA profile to found evidence may be strong circumstantial evidence, but it’s not positive identification. To better understand how often you might expect to see that profile in the general population, it is important to have a large database of profiles as a reference point.”
The current database of mitochondrial genomes available to forensic experts in the United States contains about 4,300 individuals. The new grant will allow the researchers to sequence an additional 10,000 genomes from samples collected by the Penn State Health Milton S. Hershey Medical Center.
To sequence the genomes, the researchers are using the “PacBio Sequel IIe” instrument operated by the Genomics Core Facility at the Penn State Huck Institutes of the Life Sciences. The researchers first split the mitochondrial genome into two sections of more manageable length, each of which is then copied within the machine. Each section of DNA is sequenced multiple times and multiple copies are sequenced to catch any potential errors, leading to a sequence with what researchers call “high fidelity.”
Whereas previous techniques to sequence relatively long DNA strands (long read) produced higher error rates than techniques to sequence short strands (short read), the instrument allows long-read sequencing with high fidelity—a relatively new combination.
“Traditional long-read methods have an error rate of 10%, while this method produces an error rate between 0.1 to 1%, which is comparable to short-read techniques,” said Miriam Foster, graduate student at Penn State. “This is the first time, as far as we know, that anyone has tried to use this technology to sequence so many mitochondrial genomes.”
So far, the research team has extracted DNA from more than 3,200 samples and have sequenced more than 1,500 of those.
“Because these techniques have not been done in this context before, we have spent considerable time and effort to optimize our protocols to extract and purify the DNA and prepare the DNA for sequencing,” said Jade Korber, graduate student at Penn State. “Our efforts should improve the process for any other labs who wish to use these techniques in the future.”
The researchers noted that their samples are predominately from people of European ancestry. In the future, in addition to increasing the overall quantity of samples, they said they hope to expand the database to include sequences of people from a variety of backgrounds.
“Expanding the database to be as large and as representative as possible will improve our understanding of the relative rarity of mitochondrial genome sequences as well as our ability to use these data in forensic contexts,” said Holland. “If we can access samples from additional biorepositories and secure funding, it’s reasonable that we could push this to 50,000 samples in the coming years.”
The research team also includes Jennifer McElhoe, associate research professor of biochemistry and molecular biology.