NEW YORK (GenomeWeb) − As part of a larger conservation effort, researchers from the National Oceanic and Atmospheric Administration (NOAA) have collaborated with Swift Biosciences to create DNA libraries for the critically endangered vaquita porpoise (Phocoena sinus), a small porpoise species that can only be found in the northern Gulf of California.
The biological samples, provided by NOAA's Southwest Fisheries Science Center, included tissue and extracted DNA and varied greatly in quality. Using Swift Biosciences' single-stranded DNA library kit, researchers were able to create DNA libraries from degraded samples using smaller, single-stranded fragments.
The single-stranded library kit uses a unique, sequential adaptation process to attach adapters to ends of single-stranded DNA fragments. The process includes end repairs, tailing of 3' ends, and ligation of the first truncated adapter to 3' ends; facilitating ligation of the second truncated adapter; removing oligonucleotides and small fragments; adding the second truncated adapter to 5' ends; and using PCR to increase yield and add the indexed adapter sequence.
Before they were able to successfully create a library, they first had to address the issue of sample degradation. "We had samples, but they date back mostly between 15 and 20 years ago," said Phillip Morin, molecular geneticist at NOAA. While they did have several nice extracted DNA samples, there were not many to work with, Morin explained. Although they did have tissue samples of the porpoise, many of them were not well preserved. Consequently most of them didn't have enough double-stranded fragments to successfully create a DNA library using a traditional double-stranded kit.
However, even the heavily degraded samples had single-stranded DNA fragments that could successfully create a library using a single-stranded kit. "The fragments were actually fairly long, greater than 100 base pairs." said Tim Harkins, recently appointed CEO of Swift Biosciences and collaborator on the project. Harkins said that the truly exciting part of the project was their ability to create the DNA libraries with a 100 percent success rate.
"The community as a whole struggles with quantitating DNA," said Harkins, "and the technique of quantitation varies [across the field]." He said this made the 100 percent success rate using degraded DNA samples to create DNA libraries that much more exciting to all the researchers involved.
When they were first discovered in 1958, scientists estimated that there were 1,000 vaquita porpoises. Today, there are less than 100 known individuals left in the vaquita porpoise population. Their numbers are now experiencing rapid decline — about 18 percent each year since 2011 according to the International Committee for the Recovery of the Vaquita. Scientists have predicted that if the population decline is not stopped soon, the vaquita porpoise will be extinct by 2018.
Population decline is largely due to the fisheries industry. Fisherman in the area primarily use gillnets to capture fish and shrimp to sell, but these small porpoises are often caught as well and die in the nets. The Mexican government has worked hard to push through laws to prevent illegal fishing, designate an area of the northern Gulf of California as a conservation area for the vaquita porpoise, and monitor legal fishery activities.
However, some fishermen ignore these policies. There is great demand from the Chinese market to catch another rare fish in the area called the totoaba, which shares the northern Gulf of California with the vaquita porpoise. Their swim bladders are highly prized in the natural medicine market in China and buyers will pay exorbitant prices for them — about $4,000 per pound of dried swim bladder, according to the World Wildlife Fund.
Now that researchers have successfully created DNA libraries for the vaquita porpoise, they hope to learn more about its genetics. "We can use these libraries that we have prepared to look at their genomes and try to identify the genes that make it unique to its species, for example its warm water adaptation" said Morin. The vaquita porpoise is the only living porpoise that has adapted to warm water conditions.
Researchers hope that learning more about the vaquita's genetics will help raise awareness about this unique species. Morin said that the research team's analysis already found that the vaquita porpoise is more closely related to the spectacled porpoise (Phocoena dioptrica) and the Burmeister's porpoise (Phocoena spinipinnis) from the southern hemisphere rather than the Dall's porpoise (Phocoenoides dalli) and Harbor porpoise (Phocoena phocoena), who both dwell in the North Pacific.
Scientists are also hopeful that these techniques to create DNA libraries out of degraded and damaged DNA will prove useful in genetic analysis of other rare species. "The quality [of the sample] is limited by the state of the animal at the time of collection or by the type of tissue that we are getting," said Morin. He mentioned that the collection at NOAA, while extensive, has many samples in varying states of degradation.
Many of the species that NOAA studies are, like the vaquita, endangered. Acquiring any samples at all of these animals can take decades. So, having a way to make those samples usable for analysis and sequencing is an ideal situation for them. "This is a great way to use these technologies for all sorts of different species," said Morin.