Choosing a Better Path for Your NGS Workflow

Imagine you are traveling in your car and must pass through a mountain range to get to your destination. You’ve been following a set of directions when you realize you have a decision to make. Will you stay on your current route, which is many miles shorter but contains a long tunnel that cuts straight through the mountains and obstructs your view? Or will you switch to a longer, more scenic route that bypasses the tunnel ahead and gets you to your destination a bit later than you wanted?

Choosing which route to take illustrates a clear trade-off that has to be considered—which is more valuable, speed or understanding? Yes, the tunnel gets you from one place to another faster. But what are you missing as a result? Is it worth a little extra time to see the majestic landscape that you are passing through?

Considering this trade-off is especially critical for researchers working with human DNA purified from formalin-fixed paraffin-embedded (FFPE) or circulating cell-free DNA (ccfDNA) samples for next-generation sequencing (NGS). These sample types present a few challenges when performing NGS. FFPE samples are prone to degradation, while ccfDNA samples are susceptible to gDNA contamination, and both offer a very limited amount of starting material to work with.

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Elephant Endotheliotropic Herpesvirus—A Tiny Virus Threatens the World’s Elephants

My favorite ice-breaker of all time is: “List one fact about you that no one would guess”. It is my favorite because I have an awesome answer (if I do say so myself). My go-to answer is: I spent a summer working with elephants.

It was the summer before I graduated from college, and it was really only one elephant, a five-year-old African elephant named Connie. Connie was intelligent, curious and mischievous—her favorite game with me was trying to untie my shoelaces (hint: double knotting is important). Working with her was one of the most amazing experiences of my life and left me with an abiding love for these creatures.

Elephant Endotheliotropic Herpesviruses threaten captive breeding programs. Copyright.

Understandably, I was excited last year when one of my fellow bloggers wrote about Promega helping support the work of Virginia Riddle Pearson, who was working to identify and track strains of elephant endotheliotropic herpesvirus (EEHV) in African Elephant populations. EEHV is associated with the lethal elephant hemorrhagic disease (EHD) (1). This disease is a serious threat to the captive breeding programs of these endangered creatures. Between 1962 and 2007, it accounted for 58% of the deaths of North American captive-born Asian elephants between 4 months and 15 years of age (1). These deaths include the first Asian elephant calves born at the National, Oakland and Bronx Zoos. EHD also claimed the first live-born Asian elephant calves conceived by artificial insemination in both North America and Europe. Continue reading

Better NGS Size Selection

One of the most critical parts of a Next Generation Sequencing (NGS) workflow is library preparation and nearly all NGS library preparation methods use some type of size-selective purification. This process involves removing unwanted fragment sizes that will interfere with downstream library preparation steps, sequencing or analysis.

Different applications may involve removing undesired enzymes and buffers or removal of nucleotides, primers and adapters for NGS library or PCR sample cleanup. In dual size selection methods, large and small DNA fragments are removed to ensure optimal library sizing prior to final sequencing. In all cases, accurate size selection is key to obtaining optimal downstream performance and NGS sequencing results.

Current methods and chemistries for the purposes listed above have been in use for several years; however, they are utilized at the cost of performance and ease-of-use. Many library preparation methods involve serial purifications which can result in a loss of DNA. Current methods can result in as much as 20-30% loss with each purification step. Ultimately this may necessitate greater starting material, which may not be possible with limited, precious samples, or the incorporation of more PCR cycles which can result in sequencing bias. Sample-to-sample reproducibility is a daily challenge that is also regularly cited as an area for improvement in size-selection.

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How Do I Choose the Right GoTaq® Product to Suit My Needs for EndPoint PCR?

We offer a wide array of GoTaq® DNA Polymerases, Buffers and Master Mixes, so we frequently answer questions about which product would best suit a researcher’s needs. On the product web page, you can filter the products by clicking the categories on the left hand side of the page to narrow down your search. Here are some guidelines to help you select the match that will best suit your PCR application. Continue reading