Today’s post was written by guest blogger Anupama Gopalakrishnan, Global Product Manager for the Genetic Identity group at Promega.
Next-generation sequencing (NGS), or massively parallel sequencing (MPS), is a powerful tool for genomic research. This high-throughput technology is fast and accessible—you can acquire a robust data set from a single run. While NGS systems are widely used in evolutionary biology and genetics, there is a window of opportunity for adoption of this technology in the forensic sciences.
Currently, the gold standard is capillary electrophoresis (CE)-based technologies to analyze short tandem repeats (STR). These systems continue to evolve with increasing sensitivity, robustness and inhibitor tolerance by the introduction of probabilistic genotyping in data analysis—all with a combined goal of extracting maximum identity information from low quantity challenging samples. However, obtaining profiles from these samples and the interpretation of mixture samples continue to pose challenges.
MPS systems enable simultaneous analysis of forensically relevant genetic markers to improve efficiency, capacity and resolution—with the ability to generate results on nearly 10-fold more genetic loci than the current technology. What samples would truly benefit from MPS? Mixture samples, undoubtedly. The benefit of MPS is also exemplified in cases where the samples are highly degraded or the only samples available are teeth, bones and hairs without a follicle. By adding a sequencing component to the allele length component of CE technology, MPS resolves the current greatest challenges in forensic DNA analysis—namely identifying allele sharing between contributors and PCR artifacts, such as stutter. Additionally, single nucleotide polymorphisms in flanking sequence of the repeat sequence can identify additional alleles contributing to discrimination power. For example, sequencing of Y chromosome loci can help distinguish between mixed male samples from the same paternal lineage and therefore, provide valuable information in decoding mixtures that contain more than one male contributor. Also, since MPS technology is not limited by real-estate, all primers in a MPS system can target small loci maximizing the probability of obtaining a usable profile from degraded DNA typical of challenging samples. Continue reading “Is MPS right for your forensics lab?”
Forensic analysts have long sought precision when determining time of death. While on crime scene investigation television shows, the presence of insects always seems to reveal when a person died, there are many elements to account for, and the probable date may still not be accurate. Insects arrive days after death if at all (e.g., if the body is found indoors or after burial), and the stage of insect activity is influenced by temperature, weather conditions, seasonal variation, geographic location and other factors. All this makes it difficult to estimate the postmortem interval (PMI) of a body discovered an unknown time after death. One way to make estimating PMI less subjective would be to have calibrated molecular markers that are easy to sample and are not altered by environmental variabilities.
Bacterial communities called microbiomes have been frequently in the news. The influence of these microbes encompass living creatures and the environment. Not surprisingly, research has focused on the influence of microbiomes on humans. For example, changes in gut microbiome seem to affect human health. Intriguingly, microbiomes may also be a key to determining time of death. The National Institute of Justice (NIJ) has funded several projects focused on the forensic applications of microbiomes. One focus involves the necrobiome, the community of organisms found on or around decomposing remains. These microbes could be used as an indicator of PMI when investigating human remains. Recent research published in PLOS ONE examined the bacterial communities found in human ears and noses after death and how they changed over time. The researchers were interested in developing an algorithm using the data they collected to estimate of time of death. Continue reading “Revealing Time of Death: The Microbiome Edition”
“How do you like the name Jack?” the woman on the phone asked.
On April 26, 1964, a nurse came into the hospital room of Dora Fronczak, who had just given birth to her young son, Paul. She told Mrs. Fronczak that it was time to take the baby to the nursery (at that time newborns did not stay in the room with the moms), took the baby, and left. A few hours later, another nurse came into the room to take young Paul to the nursery. It was then that everyone realized a mother’s worst fear: Her infant had been stolen.
Authorities were able to determine how the woman left the hospital and that she got into a cab, but they were never able to find the woman. However in 1965, a small toddler-aged boy was found, abandoned outside a store in New Jersey. Blood tests were not inconsistent with him being Paul Fronczak (DNA testing was not available), and there were no other missing children cases in the area that were matches. The little boy was sent to Chicago as Paul Fronczak and the case was closed.
However, as an adult Paul Fronczak, began to suspect that the couple who raised him were not his biological parents, and in 2012 Paul underwent DNA analysis to test his suspicions. The results showed that indeed, he was not the biological son of Dora and Chester Fronczak. His next step was to enlist the help of a genetic genealogist to assist him in finding his true biological parents and his identity.
By conducting “familial searches” using commercially available DNA databases like 23andMe and AncestryDNA and many resources, the genealogist’s group found a match to his DNA on the east coast. Further ground work, discovered that this family was indeed Paul’s…now Jack.
The knowledge of Jack’s true identity, didn’t bring with it a joyous union of the adoptive family who had raised and loved Jack (as Paul) with the biological family who had pined for him over the years as many might imagine. Continue reading “A Cold Case, A Mystery, and DNA”
We shared in laughter and tears. We tempered our scientific pursuit of the truth with the story of an unimaginably strong survivor of rape. We witnessed the struggles of a man trying to find his identity and the joy of being reunited with real family members after 30 years of lies. I find it hard to succinctly describe to others what my first ISHI conference was like. There is perhaps nothing more personal than our own genetic identities. This conference didn’t shy away from the raw emotions that encompass the human experience. We define ourselves as employees of this company or researchers at that institution, competing for attention and funding, yet this conference reveals how limiting these preconceptions may be.
The desire to make the world a better place unites us. I spoke with analysts for hours about the challenges of overcoming the sexual assault kit backlog, I made a fool of myself dancing to musical bingo with new friends from the Philippines and Brazil, and I was inspired by the casual musings of a video journalist. We are sure to see countless more ethical debates on how we should be using DNA (or proteins!) for human identification. The field of science relies on the open sharing and exploration of new ideas, and as admittedly biased as I am to the conveniences of the digital age, there has never been a better time to come together in person.
Here at Promega we receive some interesting requests…
Take the case of Virginia Riddle Pearson, elephant scientist. Three years ago we received an email from Pearson requesting a donation of GoTaq G2 Taq polymerase to take with her to Africa for her field work on elephant herpesvirus. Working out of her portable field lab (a tent) in South Africa and Botswana, she needed a polymerase she could count on to perform reliably after being transported for several days (on her lap) at room temperature. Through the joint effort of her regional sales representative in New Jersey/Pennsylvania (Pearson’s lab was based out of Princeton University at the time) and our Genomics product marketing team, she received the G2 Taq she needed to take to Africa. There she was able to conduct her experiments, leading to productive results and the opportunity to continue pursuing her work. Continue reading “Of Elephant Research and Wildlife Crime – Molecular Tools that Matter”
Forensic lab validations can be intimidating, so Promega Technical Services Support and Validation teams shared these tips for making the process go more smoothly.
Prepare Your Lab. Make sure all of your all of your instrumentation (CEs, thermal cyclers, 7500s, centrifuges) and tools (pipettes, heat blocks) requiring calibration or maintenance are up to date.
Start with Fresh Reagents. Ensure you have all required reagents and that they are fresh before beginning your validation. This not only includes the chemistry being validated, but any preprocessing reagents or secondary reagents like, polymer, buffers, TE-4 or H2O.
Develop a Plan. Before beginning a validation, take the time to create plate maps, calculate required reagent volumes, etc. This up-front planning may take some time initially, but will greatly improve your efficiency during testing.
Create an Agenda. After a plan is developed, work through that plan and determine how and when samples will be created and run. Creating an agenda will hold you to a schedule for getting the testing done.
Determine the Number of Samples Needed to Complete Your Validation. Look at your plan and see where samples can be used more than once. The more a sample can be used, the less manipulation done to the sample and the more efficient you become.
Select the Proper Samples for Your Validation. Samples should include those you know you’ll obtain results with be similar to the ones you’ll most likely be using, and your test samples should contain plenty of heterozygotes. When you are establishing important analysis parameters, like thresholds, poor sample choice may cause more problems and require troubleshooting after the chemistry is brought on-line.
Perform a Fresh Quantitation of Your Samples. This will ensure the correct dilutions are prepared. Extracts that have been sitting for a long time may have evaporated or contain condensation, resulting in a different concentration than when first quantitated.
Stay Organized. Keep the data generated in well-organized folders. Validations can contain a lot of samples, and keeping those data organized will help during the interpretation and report writing phase.
Determine the Questions to Be Answered. While writing the report, determine the questions each study requires to be answered. Determining what specifically is required for each study will prevent you from calculating unnecessary data. Do you need to calculate allele sizes of your reproducibility study samples when you showed precision with your ladder samples?
Have fun! Remember, validations are not scary when approached in a methodical and logical fashion. You have been chosen to thoroughly test something that everyone in your laboratory will soon be using. Take pride in that responsibility and enjoy it.
Need more information about validation of DNA-typing products in the forensic laboratory? Check out the validation resources on the Promega web site for more information for the steps required to adopt a new product in your laboratory and the recommended steps that can help make your validation efforts less burdensome.
That is the prevailing question I’m asked when someone learns of my occupation as Deputy Sheriff Criminalist for the Contra Costa County (CA) Office of the Sheriff. Alas, my life is not quite so glamorous. It actually often entails entering formulas into an excel spreadsheet while being placed on hold as I order some pipette tips.
But, why does it have to be that way?
I have attended my fair share of professional conferences and workshops and written numerous journal articles. As a forensic scientist I do believe in the importance of sharing data, new techniques, and new methodologies with my colleagues. Yet what I think is not highlighted enough is the one element that differentiates our field from any other scientific field—our involvement with the criminal justice system. Every case we work on involves a mystery, a crime, a victim(s), and a suspect(s). And while scientists in other fields typically only speak to other scientists, in my world, forensic scientists usually interact with a person in a black robe who has the power to strongly influence the outcome of a case. These wildly frustrating, invigorating, and challenging cases are the most interesting things about our field, and yet we hardly share our stories.
The American Academy of Forensic Sciences’ 68th annual conference took place in Las Vegas February 22–27th, and those of you who did not attend, like me, had to live vicariously through the social media posts of those who did. The question on everyone’s mind: Who was up five hundy by midnight?
Okay, okay, most people who went to AAFS went for scientific purposes, and in fact, @andycyim was the only one to post a tribute to Swingers with a #vegasbaby tweet. Tip of the hat to you, Andy. So what did the Twitterverse look like during the week of the conference? I analyzed nearly 600 tweets and found some interesting patterns of how scientists interact on social media during a conference. More on my methodology is at the end of this article.
Mitochondria, often thought of as powerhouses of the cell, are fascinating eukaryotic organelles with a double-layered membrane and their own genome. Mitochondrial DNA (Mt DNA) is typically about 16570 bases, circular, highly compact, haploid and contains 37 genes, all of which are essential for normal mitochondrial function. Thirteen of these genes provide instructions for making enzymes involved in oxidative phosphorylation, a process that uses oxygen and simple sugars to create adenosine triphosphate (ATP), the cell’s main energy currency. The remaining genes code for transfer RNA (tRNA) and ribosomal RNA (rRNA) which are necessary for translating messenger RNA transcribed from nuclear DNA, into protein molecules.
Next-generation sequencing (NGS), also known as massively parallel sequencing, is revolutionizing genomic research. NGS technologies have made whole genome sequencing fast and easy, leading to dramatic advances in evolutionary biology and phylogenetics, personalized medicine and forensic science. Why is NGS such a hot topic right now?