Scientists sequence plant DNA in the field to identify species within hours

Scientists can now rapidly read the DNA of an organism—even a plant—anywhere.

Researchers at the Royal Botanic Gardens, Kew, have recently reported on their use of a handheld real-time DNA sequencing device that allowed them to identify the various species of an entire field of plants far faster than could be done using previous methods.

This was the first time genomic sequencing of plants has been performed in the field. They highlight the new opportunities that real-time nanopore sequencing (RTnS) offers for plant research and conservation.

Speeding the process of species identification

Identifying similar-looking plants has traditionally been difficult and time-consuming, requiring plant samples to be collected in the field, transported to a lab, and analyzed with large, expensive equipment, a process that took weeks or months. The lengthy process limits how fast decisions can be made regarding species newly found in forest corridors or found in international shipments of plant or animal products.

Reading the genetic code of a plant or an animal is regarded to be the most reliable way to identify its species. New DNA analysis technology has decreased the time and cost of identifying the species of a given organism. Technology to sequence DNA, which is used to determine the order of four base nucleotides—adenine, guanine, cytosine, and thymine—in a strand of DNA, has improved dramatically over the past decade. This order, or sequence, of these building blocks of an organism’s DNA is unique to each organism, so knowing the order of the DNA of a sample allows scientists to identify its species.

DNA barcoding—the collection, extraction, sequencing, and translation of a species’ DNA into a unique digital barcode—is increasingly being used to identify species, assess composition of natural communities, and combat poaching and illegal wildlife trade. Research teams are testing applications of DNA barcoding for identifying closely related animal species, such as sharks and rays, as trade in some, but not all, species is illegal. Efforts are also ongoing to apply the technology to plant products that have been processed, such as timber.

The advantage of barcoding is its ability to read short strands of DNA, which is often what are  available to scientists analyzing environmental DNA (eDNA) from soil, water, or fecal samples. To compare species, the technology requires a recoverable segment of DNA that can serve as an identification marker across species. Scientists have identified the Cytochrome C Oxidase 1 or CO1 gene region of DNA as universally usable for animal species. However, the C01 gene region evolves too slowly in plants, so scientists are still searching for the single best gene region for plants. This has limited species identification of plants in the field.

Improving the precision of species identification

Real-time nanopore sequencing (RTnS)—a relatively new DNA sequencing method—may enable rapid species identification at a relatively low cost and with minimal equipment. Nanopore sequencers add to the utility of barcoding because they can read longer strands of DNA with less preparation, such as PCR amplification or chemical labeling, of each sample.

Streamlining preparation makes the technology potentially cheaper, faster, and smaller than other DNA sequencers and, thus, useful for monitoring disease outbreaks, environmental changing, food safety, and antibiotic resistance. It also facilitates the sequencing of whole human, animal, and plant genomes.

The capacity to read longer DNA strands gives the nanopore sequencer more genetic information to work with, which enables it to more precisely distinguish samples and thus more accurately identify their species.

The Kew scientists used the MinION, a portable RTnS DNA sequencer from Oxford Nanopore Technologies, to analyze plant species in the U.K.’s Snowdonia National Park. This was the first time genomic sequencing of plants has been performed in the field.

The MinION runs off a USB cable attached to a laptop and is small enough to be portable for fieldwork. A tiny sample of blood, feces, plant tissue, or other sample is mixed with reagents and placed into the handheld device. It generates data about the sample’s DNA within minutes, which is a big advantage for investigating wildlife crimes and identifying the species of traded wildlife and plant products.

MinION has been used in field surveys to sequence animal DNA to identify species, but plant species identification through DNA has been more difficult.

“Identifying species correctly based on what they look like can be really tricky and needs expertise to be done well,” said Alexander Papadopulos, Kew scientist and co-author on the paper. “This is especially true for plants when they aren’t in flower or when they have been processed into a product.”

The research team achieved its aim of identifying and distinguishing between two closely-related, similar-looking plant species of the genus Arabidopsis—Arabidopsis thaliana and A. lyrata—in a single day of collecting and sequencing samples entirely in the field.

Adding flexibility to species identification

The new RTnS technology allowed the team to sequence random parts of the plants’ genomes, which avoids the time-consuming process of targeting specific regions of DNA, the more traditional approach for identifying species with DNA.

“Our experiments show that by sequencing random pieces of the genome in the field, it’s possible to get very accurate species identification within a few hours of collecting a specimen,” said Papadopulos in a media release. “More traditional methods need a lot of lab equipment and have often only provided enough information to identify a sample to the genus level.”

The researchers compared their field-generated DNA sequences to a freely available database of reference genome sequences, created using traditional methods, to make their identification. Once confirmed, the field-sequenced data can then themselves be used as reference samples for DNA sequences to help future identifications of those species in the field.

In their paper, the authors state, “Our analyses demonstrate that correctly identifying unknown reads from matches to a reference database with RTnS reads enables rapid and confident species identification.”

Lead author of the paper Joe Parker said in a media release, “This research proves that we can now rapidly read the DNA sequence of an organism to identify it with minimum equipment. Rapidly reading DNA anywhere, at will, should become a routine step in many research fields. Despite hundreds of years of taxonomic research, it is still not always easy to work out which species a plant belongs to just by looking at it. Few people could correctly identify all the species in their own gardens.”

Reference

Parker, J., Helmstetter, A. J., Devey, D., Wilkinson, T., & Papadopulos, A. S. (2017). Field-based species identification of closely-related plants using real-time nanopore sequencing. Scientific reports, 7(1), 8345. https://www.nature.com/articles/s41598-017-08461-5