Researchers at the University of Washington (UW), and at Microsoft, have developed a DNA-based molecular tagging system, called Porcupine, which they say could offer a cost-effective alternative to bulky plastic or printed barcodes that are commonly used in, for example, retail or manufacturing. The new molecular tags are built from dehydrated strands of synthetic DNA, which can be programmed and read within seconds using a portable nanopore device. The scientists say that as well as its use for tagging physical objects, the new system could also be used for molecular-level tagging applications, such as sample multiplexing for nanopore sequencing.
“Molecular tagging is not a new idea, but existing methods are still complicated and require access to a lab, which rules out many real-world scenarios,” said Kathryn Doroschak, a UW doctoral student in the Paul G. Allen School of Computer Science & Engineering. “We designed the first portable, end-to-end molecular tagging system that enables rapid, on-demand encoding and decoding at scale, and which is more accessible than existing molecular tagging methods.” Doroschak is first author of the team’s published paper in Nature Communications, which is titled, “Rapid and robust assembly and decoding of molecular tags with DNA-based nanopore signatures.”
The plastic tags that you might find on clothes in a store is one example of radio frequency identification technology (RFID), which, together with QR codes, and scannable barcodes, is now a commonly used method for object tracking and inventory. RFID tags have become a mainstay not just in retail, but also in manufacturing, logistics, transportation, health care and more, for example.
However, despite their widespread use, these existing object tagging systems are not always ideal, and may be easily damaged or removed. “… these tags cannot be applied to objects that are too small, flexible, or numerous, or in scenarios where the code should be invisible to the naked eye, like anti-forgery,” the scientists further explained.
While more recent advances in DNA-based data storage and computation offer new possibilities for creating a tagging system that is smaller and lighter than conventional methods, there have still been hurdles to overcome. “No molecular tagging method exists that is inexpensive, fast and reliable to decode, and usable in minimal resource environments to create or read tags,” the authors continued. “ … existing methods for encoding digital information in molecules; including tracers, silica-encapsulated DNA DNA embedded in 3D printed material, barcodes, microbial barcodes, or spatially isolated marker peptides; require access to specialized labs and equipment to make new tags—making them impractical in applications that require a very large number of tags.”
Taking advantage of recent developments in DNA sequencing technologies and raw signal processing tools, the new Porcupine technology inexpensive and user-friendly design forgoes the need for access to specialized labs and equipment, and allows dehydrated strands of synthetic DNA to take the place of bulky plastic or printed barcodes. Instead of radio waves or printed lines, the Porcupine tagging scheme relies on a set of distinct DNA strands called molecular bits, or molbits, which incorporate highly separable nanopore signals to ease later readout. Each individual molbit comprises one of 96 unique barcode sequences combined with a longer DNA fragment selected from a set of predetermined sequence lengths. Under the Porcupine system, the binary zeros and ones of a digital tag are signified by the presence or absence of each of the 96 molbits.
“We wanted to prove the concept while achieving a high rate of accuracy, hence the initial 96 barcodes, but we intentionally designed our system to be modular and extensible,” said co-author Karin Strauss, senior principal research manager at Microsoft Research and affiliate professor in the Allen School. “With these initial barcodes, Porcupine can produce roughly 4.2 billion unique tags using basic laboratory equipment without compromising reliability upon readout.”
Porcupine gets around the typically high costs associated with reading and writing DNA, by prefabricating the fragments of DNA. “Although DNA is typically considered expensive for reading and writing, Porcupine lowers the cost by presynthesizing the DNA, which can then be mixed arbitrarily to create new molecular tags,” the authors noted. “Molecular tags are then read out quickly using a portable, low-cost sequencing device.”
In addition to lowering the cost, the new approach has the added advantage of enabling users to arbitrarily mix existing strands to quickly and easily create new tags. The molbits are prepared for readout during initial tag assembly and then dehydrated to extend the shelf life of the tags. This approach has a number of advantages, the team continued. “Molbits are prepared for readout (sequencing) prior to tag application and can be stabilized by dehydration, an approach that extends tag shelf life, decreases decoding time, and reduces contamination from environmental DNA.”
Another advantage of the Porcupine system is that the tiny size of the molbits – measuring only a few hundred nanometers in length – means that each molecular tag is small enough to fit over a billion copies within one square millimeter of an object’s surface. This makes them ideal for keeping tabs on small items or on flexible surfaces that aren’t suited to conventional tagging methods. Invisible to the naked eye, the nanoscale form factor also adds another layer of security compared to conventional tags.
“Unlike existing inventory control methods, DNA tags can’t be detected by sight or touch, said senior author Jeff Nivala, PhD, a research scientist at the Allen School. “Practically speaking, this means they are difficult to tamper with. This makes them ideal for tracking high-value items and separating legitimate goods from forgeries. A system like Porcupine could also be used to track important documents. For example, you could envision molecular tagging being used to track voters’ ballots and prevent tampering in future elections.”
To read the data in a Porcupine tag, a user rehydrates the tag and runs it through a portable nanopore device. To demonstrate, the researchers encoded and then decoded their lab acronym, “M-I-S-L,” reliably and within a few seconds using the Porcupine system. “In summary, Porcupine offers a method for molecular tagging based on the presence or absence of synthetic DNA sequences that generate explicitly unique nanopore raw current traces,” the team noted. “By directly manipulating segments of nanopore raw current and keeping unique sequences short, we reduce synthesis costs for the end user and produce visually unique nanopore current traces, enabling high accuracy decoding.”
Co-author Luis Ceze, PhD, a professor in the Allen School, further commented, “Porcupine is one more exciting example of a hybrid molecular-electronic system, combining molecular engineering, new sensing technology and machine learning to enable new applications.” And as advancements in nanopore technologies make these systems progressively even more affordable, the team believes molecular tagging could become an increasingly attractive option in a variety of real-world settings.