STR-Validator: Open Source, Free Software for Evaluating Validation Data in the Forensic Laboratory

Before an established method or procedure can be employed in a forensic laboratory, an internal validation must be completed to show that the method performs as expected. Guidelines for validation are outlined by the Scientific Working Group on DNA Analysis Methods (SWGDAM) and the European Network of Forensic Science Institutes (ENFSI) DNA Working Group. Validation experiments that meet these guidelines will demonstrate the sensitivity and reliability of a short tandem repeat (STR) typing multiplex system. After a lab completes these validation experiments, it will have sufficient data to determine the analytical and stochastic thresholds of the capillary electrophoresis (CE) instrument in combination with the amplification system, the impact of multiple contributors to a DNA sample and the limit of detection and accuracy of the assay.

Such forensic lab validations are time consuming and can be intimidating, and the requirement to validate new technologies and systems is often seen as a deterrent to the adoption of new technologies or improved chemistries in a forensics laboratories. Any tools or tips that can reduce the barrier of validation, may also help the field of DNA forensics implement new technologies more quickly.

On October 1, Oskar Hansson, from the Department of Forensic Medical Services at Oslo University Hospital, will be leading a workshop entitled “Efficient Validation Using STR-Validator” as part of ISHI 28. This workshop introduces the free, open-source STR-Validator software tool that is designed to assist forensic laboratories in the evaluation of validation data. STR-validator is a free and open source R-package developed mainly for internal validation of forensic STR DNA typing kit. However, it is equally suited for validation of other methods and instruments, or for process control. The graphical user interface of the software enables easy analysis of data exported from software programs like GeneMapper® software, without any knowledge about R commands. The software also provides convenient functions to import, view, edit, and export data. After completed analysis, the results, plots, heat-maps, and data can be saved for easy access. Currently, analysis modules for stutter, balance, drop-out, concordance, mixtures, precision, pull-up, result types, and analytical threshold are available. STR-validator can greatly increase the speed of validation by reducing the time and effort needed for analysis of the validation data.

The workshop will include lectures and demonstrations to introduce STR-Validator as an efficient tool for the analysis of validation data in accordance with ENFSI recommendations and SWGDAM guidelines. This workshop is suitable for DNA analysts, technicians and QA/QC managers.

Have you registered for ISHI 28 in Seattle? Check it out. This year’s panel discussion will take up the topic of familial searching. Preregister for workshops. Read speaker bios.

Interested in more tips for smoother validation in your lab? This blog has several suggestions.

Optimizing tryptic digestions for analysis of protein:protein interactions by mass spec

Protein:protein interactions (PPIs) play a key role in regulating cellular activities including DNA replication, transcription,translation, RNA splicing, protein secretion, cell cycle control and signal transduction. A comprehensive method is needed to identify the PPIs before the significance of the protein:protein interactions can be characterized. Affinity purification−mass spectrometry (AP−MS) has become the method of choice for discovering PPIs under native conditions. This method uses affinity purification of proteins under native conditions to preserve PPIs. Using this method, the protein complexes are captured by antibodies specific for the bait proteins or for tags that were introduced on the bait proteins and pulled down onto immobilized protein A/G beads. The complexes are further digested into peptides with trypsin. The protein interactors of the bait proteins are identified by quantification of the tryptic peptides via mass spectrometry.

The success of AP-MS depends on the efficiency of trypsin digestion and the recovery of the tryptic peptides for MS analysis. Several different protocols have been used for trypsin digestion of protein complexes in AP-MS studies, but no systematic studies have been conducted on the impact of trypsin digestion conditions on the identification of PPIs.  A recent publication used NFB/RelA and BRD4 as bait proteins and five different trypsin digestion conditions (two using “on beads” and three using “elution” digestion protocols). Although the performance of the trypsin digestion protocols changed slightly depending on the different bait proteins, antibodies and cell lines used, the authors of the paper found that elution digestion methods consistently outperformed on-beads digestion methods.

Reference

Zhang, Y. et al. (2017) Quantitative Assessment of the Effects of Trypsin Digestion Methods on Affinity Purification−Mass Spectrometry-based Protein−Protein Interaction Analysis
J of Proteome. Res. 16, 3068–82.

Predicting the Future with Dirty Diapers

Microbiome research is booming right now, with more and more evidence that our personal health and environment are shaped and influenced by the microbes we harbor and encounter. One area of study I find particularly interesting is how the microbiome we acquire at birth affects our long-term health.

A flood of new findings have emerged related to infant microbiome research, leaving parents like me scratching their heads about whether the secrets to our children’s future health may exist in the seemingly endless stream of dirty diapers we change.

The human microbiome evolves and develops in utero and then during and after delivery is colonized by bacteria encountered during exposure to the external environment. The initial composition of microbes an infant is populated with influences their lifelong microbiome signature and can be influenced by many factors along the way, including the microbiome community of the mother, use of antibiotics or other antibacterial substances, breastfeeding, C-section birth. These variables have been correlated with disruption of the infant microbiome and associated with differences in cognitive development and the development of disease, such as asthma and allergies.

In general, these correlations are discovered by taking a fecal sample from an infant and analyzing the DNA sequences of the bacteria present. The microbiome composition of the individual is then compared against different individual characteristics (such as presence or absence of a disease) at the time of the sample and/or at later points in time. Finally researchers look for statistically significant patterns among individuals with similar characteristics or microbiome communities. This type of study can reveal associations between the microbiome and individual traits, but further experiments are needed to show causation. Continue reading

“Reverse” Molecular Reactions in DNA through Mind-Body Interventions

While my morning routine typically only involves a large cup of coffee, increasingly more Americans are beginning their days with a set of sun salutations. Sun salutations are a series of poses originating from yoga, one of the most popular types of mind-body intervention in the United States. Along with yoga, other commonly recognized mind-body interventions (MBI) include meditation, mindfulness, Tai chi, and Qigong. Despite the fact that each of these activities differ in the amount of physical effort required, they all view mental and physical health as single cohesive system.

The influence of overall mind-body intervention on health and wellness is an ancient concept that is now revolutionizing Western medicine. In the past, Western medicine has focused primarily on the health of the physical body. Yoga and meditation were viewed as beneficial, but were less likely to be recommended by clinicians as a method for relief. Now, with recent developments in gene expression analysis techniques, we have a better understanding of biological mechanisms and how they interact with psychological variables. A possible shift in clinician’s philosophies can be seen in the steady rise in the complementary health approaches of yoga, Tai chi, and qi gong1.

To completely understand how MBI affects a person’s health, we must first realize the links between stress and the conserved transcriptional response to adversity (CTRA). CTRA refers to the common molecular pattern discovered in individuals facing hardship. Whether it be in the form of diagnosis of a life-threatening disease or the death of a loved one, the characteristics of CTRA stay consistent. CTRA causes an influx in the production of epinephrine and norepinephrine. These neuromodulators then affect the production of transcription factors. Continue reading

Five Summer Science Projects that are so Fun Your Kids Won’t Realize They are Learning

It is summer here in Wisconsin and the kids are out of school. If you are like me, you are looking for things to keep them busy and (bonus!) maybe teach them something. Below is a list of relatively easy, do-at-home science projects that can be fun for the whole family to try.

Parental supervision is recommended/required for these. And if you don’t want to worry about major clean up (or repainting walls and ceilings) you might want to do these outside whenever possible. I might be speaking from personal experience on this point, so trust me.

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An Unexpected History Lesson

Hillside Trail, Muir Woods National Monument

Hiking the Hillside Trail in Muir Woods National Monument

This land is your land, this land is my land
From California to the New York Island
From the Redwood Forest, to the Gulf stream waters
This land was made for you and me

–Woodie Guthrie

When my daughter was in preschool, she learned the lyrics to Woodie Guthrie’s folk song This Land Is Your Land. After one summer vacation, while she played in the Gulf stream waters off the coast of Florida, she asked, “Can we go see the Redwood Forest now?”

I had never seen the Redwood Forest, and my daughter’s request piqued my curiosity. I thought about my own childhood, when I had accompanied my older sister on a botany class project to collect plants and how curious I was about the plants and where they grew and what their names meant. Suddenly I wanted to see the Redwoods, and the Giant Sequoias.

It took a few years, but we managed to design a vacation trip that satisfied my daughter’s request to see the Redwood Forest and my growing curiosity, and I am so glad we did.  Continue reading

First We Eat: #LifeatPromega, The Culinary Experience

The Promega Culinary Garden

In the Promega garden. (L to R): Logan Morrow, gardener; Nate Herndon, Promega Senior Culinary Manager; Mike Daugherty, Promega Line Cook and Gardener

First we eat, then we do everything else.–MFK Fisher

Swatting away mosquitoes one July morning in the garden on the Promega Madison, WI, campus, Senior Culinary Manager Nate Herndon leans down and pulls back the leaves of a squash plant, revealing the bright yellow flowers that in a couple of hours will highlight a seasonal special on the lunch menu at one of the company’s cafeterias: green onion-cream cheese stuffed fried squash blossoms served on a grilled jerk pork tostada with black beans and cilantro sauce. Herndon explains that dishes made from scratch with high-quality, locally sourced (and sometimes unexpected) ingredients are the rule at Promega Madison kitchens, where it’s not uncommon to find entrees like house made ramp garganelli with oyster mushrooms and asparagus, braised beef ragu with house made buckwheat parpadelle pasta and baby kale, or fried perch tacos.

Food is an extension, a daily demonstration, of our overall commitment to sustainability, the community and employees

Many companies are realizing the benefits of upscaling their corporate cafeteria offerings. Some are engaging employees with ever-changing theme lunch menus or energy drinks on tap. Others are echoing the popular farm to table movement. But Herndon explains that Promega’s sensibilities surrounding the importance of food goes way beyond simply following popular trends. Continue reading

Helping Others through Science and Service

Science has been an important part of my life for a long time. One of my motivations for being a scientist was to help people. As scientists, there are many ways that we make a difference. For example, doing research that reveals information about basic biological processes can provide insight into how a disease might wreak havoc, and in turn facilitate drug design and effective disease treatments. I can say from experience that it’s especially rewarding to go beyond the impact of science to assist someone in the community face to face.

A St. Vincent de Paul Food Pantry host helps a client to shop for food.

Just over 5 years ago, I started volunteering at the St. Vincent de Paul Madison Food Pantry, the largest in Dane County, Wisconsin, which serves an average of about 400 families per week1. The pantry uses a customer-choice model in which clients are allotted points to shop for food, allowing them to make selections that preserve their dignity and ethnic diversity. The food pantry has a small staff, so volunteers are vital to keep things running. I serve as a “host” to clients and assist them to shop around the pantry for the items that they need. It has been such a positive experience for me. In the grand scheme of things, I’m not changing the world, but I’m helping someone to get essential items to make ends meet for their family. Tough times can happen to anyone, and it takes a great deal of courage to ask for help. My goal is to make the experience for clients as positive as possible by being cheerful, courteous and respectful during their time at the pantry. If my help can make a person forget even for a moment that they have fallen on hard times, then I call that a win!

A desire to make a difference in the community through volunteerism is one of the characteristics that I really like about working at Promega. At a recent company meeting, employees were asked to share how they serve the community. Activities ranged from assisting those with disabilities to participate in athletic activities to taking care of shelter animals to starting a non-profit for children in need. There were many more! Employees helped those in their local communities and even those across the globe from where they live. It was so inspiring to hear about my colleagues’ experiences of serving others.

Promega has a mechanism for employees to apply for time off to volunteer through the Promega in Action program. Continue reading

Postcards from the Northern Roman Empire

Some of the thin wood tablets found at Vindolanda in Northumberland, England. Image Copyright The Vindolanda Trust.

Correspondence whether via postcard or letter has been a method of human communication likely since people became literate. Old letters and postcards have been uncovered in attics, basements and garages, offering depth and richness to historical events or adding context to how humans lived in the past. But what about finding correspondence from more than a few hundred years ago?

Interestingly, archeologists were excavating in a Roman fort just south of Hadrian’s Wall and discovered well-preserved thin slices of wood with ink writing dating to the 1st century. While these 25 postcard-sized correspondence, found in a line about 3–4 meters long, are just the latest uncovered at the Vindolanda fort, the documents add to the history of Romans in Britain.

Many of the newly discovered wooden wafer postcards seemed to contain complete messages and could be read without the need for infrared photography. This treasure “hoard” of ancient Roman writing tablets offer insights such as a man named Masclus asking for leave. His previously discovered correspondence also from the Roman fort at Vindolanda included asking his commanding officer to supply more beer to his outpost.

The announcement of these 25 new Roman messages by the sponsors of the fort excavation are only preliminary overview of the find. In fact, archeologists are working on conserving and deciphering messages on the wooden tablets and plan on using infrared photography to reveal if there is any more writing on these postcards from the past.

Read more in the Vindolanda Trust Press Release.

Promega Third Party Forensic-Grade Certification

Promega has become the first major forensic manufacturer to achieve third party certification of the published ISO 18385 standard to minimize the risk of human DNA contamination in products used to collect, store and analyze biological material for forensic purposes.

On February 2, 2016, ISO 18385:2016 was published as the first international standard specific to the forensic manufacturing community. Since the standard was published, companies have begun to self-declare that they comply with the ISO standard. Some companies have gone a step further and reached out to Certification Bodies to provide an unbiased and independent assessment their compliance to ISO18385 through a third-party audit.

When consumers see an ‘ISO 18385 Forensic Grade’ labeled product, it should inspire confidence that the product was produced in accordance with a minimum set of criteria common to all manufacturers.

So what are you actually getting in a Forensic Grade labeled product? Continue reading