A Sketch For Success

The problem

You’ve just published a paper in a highly ranked cancer research journal. It’s a project that has taken years of your life and hundreds of thousands of dollars in funding. It has been challenging, exhausting, and exciting – and it’s just the first step on the road to a much broader answer; maybe even a cure.

But there’s a problem. Another laboratory, attempting to reproduce your results, hasn’t been able to. Why? Extensive testing points the finger at the cell line you used in your experiments – a cell line you use in almost all of your work. Suddenly, you’re faced with questions: Is that cell line really what you think it is, genetically? Can you trust it? And if not, are your results still meaningful?

Background

Cell line authentication is a vital part of medical research – and yet, because of the time and effort required by current methods, it’s often postponed or overlooked. This, and many other problems across research and clinical boundaries, could be solved by the application of a rapid DNA re-identification method. That’s why my colleagues and I developed MinION sketching (1) – a new way of using existing technology to make DNA identification fast, cheap and manageable.

It was the idea of a portable DNA sequencer that really triggered our imagination. The ability to take a DNA sequencer anywhere to analyze nature outside the walls of a specialized laboratory is a true game changer. Imagine being able to re-identify individuals at crime scenes almost in real time – using this knowledge to prevent a perpetrator from striking again. DNA fingerprinting currently takes days, between sample transport, queuing, preparation, and running the DNA sequencing devices and interpretation software. A portable DNA sequencer would solve this problem, we thought, allowing us to re-identify DNA samples on-site.

While working on developing robust methods to employ portable DNA sequencers in the field, we realized that the method we had devised (see “MinION Sketching”) could also make a difference to a long-standing problem: periodic cell line authentication in research labs. Cell lines derived from patients are crucial for the research of specific diseases. In the lab, such cells are carefully studied to understand the molecular mechanisms behind the illness. Although each cell line behaves a little differently on a molecular level, they often look very similar under the microscope. As a result, accidental contamination, mislabeling or label swapping is an unfortunate and hard-to-track inevitability; for scientists who may spend years figuring out the underlying molecular mechanisms of a particular diseased cell line, that can lead to disaster.

The solution

The lack of cell line authentication is a long-standing problem in disease research and, because it results in irreproducible research, a major cause of wasted research money. However, the problem is not because of an absence of available tools; rather, it’s because of the time and effort it takes to use those tools. Our method enables rapid, on-site identification via DNA fingerprinting, an approach borrowed from the forensic sciences as an excellent method to help authenticate the origins of cell lines. The methods currently offered by third parties or done in-house are long and tedious; our method, in contrast, allows rapid checking by DNA fingerprinting as part of the standard laboratory toolkit. And that could reduce scientists’ resistance to regular testing. Not only will this be a step toward making research with cell lines more efficient, but it will also eventually bring us closer to cures for a multitude of diseases.

When we started our project, the MinION portable DNA sequencer had a high reading error rate. For approximately every 10 nucleotides it read, one was wrong. The difference between individuals is about one in every 1,000 nucleotides – so a 10 percent error rate was unacceptable! We needed to find a way to bridge the gap and still be able to use some of the MinION data. To that end, we developed a weighing method in which we determine the probability that a given nucleotide reading is an error and then consider the probability that we might see it in the general population. Of course, the less commonly observed a nucleotide is in the population, the more informative it will be in attempting to trace a sample back to a single individual.

The MinION reads DNA in real time, so each informative nucleotide that comes off the DNA sequencer is another piece of evidence. The evidence sequentially updates the posterior probability of a match to one reference file in the database. If the MinION sketch does not match any entry in the database, all posterior probabilities will stay low and the method won’t yield a match – but if the probability of a match is high, the method will flag that file in the database for review. We have tested extensively for false positives and optimized our method so that we don’t run into such problems. The re-identification opportunity is only as good as the database, of course – as with all forensic methods – but if the database doesn’t contain a corresponding reference file, there will be no match.

Beyond the solution

The technique has a number of applications, both within and beyond the walls of the laboratory.

Basic research

Cell line authentication isn’t the only use for MinION sketching. Because it doesn’t selectively amplify specific stretches of DNA like other methods, it allows for the identification of pathogens infecting those lines. For instance, Mycoplasma contamination often affects laboratory cell lines and can be challenging to detect.

Forensic science

Our method can be used as a tool for rapid re-identification of individuals after mass disasters. After such events, family members are understandably keen to locate their loved ones. They can contribute by sharing their SNP reference files with the forensic team to facilitate matching and re-identification analyses. The samples can rapidly be checked on-site, even in remote areas, letting families be reunited with their missing members – an amazing advancement. 2017 was the 20-year anniversary of GATTACA – a movie that predicts a future in which identities are verified not by cards or photographs, but by DNA fingerprint matching. With MinION sketching, such a future may not be far off. Will we soon give up our passports in favor of our DNA?

In the clinic

DNA fingerprinting is an easy way to track clinical samples. It can allow the identification of infectious pathogens, too, as well as any antimicrobial resistance markers that might affect treatment decisions (2). I also foresee its application in organ transplantation verification; immediately before surgery, the patient and the donor organ can rapidly be authenticated as a last check for a correct donor-recipient match.

There are many opportunities to use our method in other fields. For instance, we have investigated its use in dog and cow re-identification. This enables high-resolution tracking of individual species. Instead of a microchip system, owners can be reunited with their lost dogs via a quick DNA test. Cows can potentially be traced from farm to table. If we can do that, what about tracking high-value horses? Cats? Animals at risk of poaching (2)? The list is endless!

The best part? In many of these examples, it’s not a “blue-sky future” projection – it’s already here. People can begin using MinION sketching right now! For cell line authentication, for instance, the first task is to compile a good reference database of all cell lines present in the laboratory. Then, it’s just a matter of a few easy steps:

• Set up of our Person ID pipeline on a computer (see “MinION Sketching”). This requires some command-line knowledge. 
• In the wet lab, extract DNA from each cell line.
• Perform a MinION library preparation of the DNA.
• Sequence the DNA and generate a MinION sketch.
• Run the Person ID pipeline and analyze the matching results against your compiled database.

MinION sketching is a “new kid on the block” for a vast number of applications. My colleagues and I are still working to develop it, of course, but it’s already available for use – and it’s a simpler, cheaper alternative to many of the current options. It’s my hope that, as both the technology and the availability of reference genomic data advance, researchers and laboratory medicine professionals will be able to work with confidence, knowing that their cell line – and their science – is trustworthy.

Sophie Zaaijer is CEO and co-founder of PlayDNA, and Runway Startup Postdoc at the Jacobs Technion-Cornell Institute, Cornell Tec, New York, USA.