NEW YORK (GenomeWeb) – Museum specimens may contain information that could be useful to biologists, but can be intractable for DNA sequencing because of their age or their preservation in fluid.
To address this problem, and possibly open the variety of samples available in museums worldwide to scientists, two researchers from Rutgers University and Louisiana State University devised a modified DNA extraction protocol and combined it with high-throughput sequencing. As they reported in Molecular Ecology Resources this week, they were able to recover DNA from formalin-fixed and fluid-preserved snakes that were collected more than 100 years ago.
"We successfully extracted DNA and sequenced ultraconserved elements (2,318 loci) from 10 fluid-preserved snakes and included them in a phylogeny with modern samples," wrote Rutgers' Sara Ruane and LSU's Christopher Austin. "This phylogeny demonstrates the general use of such specimens in phylogenomic studies and provides evidence for the placement of enigmatic snakes, such as the rare and never-before sequenced Indian Xylophis stenorhynchus."
Ruane and Austin started by using the web portals VertNet, IUCN Redlist, and the Reptile Database to compile a list of 21 rare snakes from 17 species that had been obtained by various museums from the 1870s to 1990, and borrowed liver tissue samples. Sixteen of the specimens were formalin-fixed, but others were fixed with alcohol.
After testing different extraction methods, they settled on the Qiagen DNeasy Kit. Extracts with quantifiable DNA and some negative controls were sent to MYcroarray for ultra-conserved elements (UCE) library preparation using the MYbaits tetrapod 5K kit, and were then sequenced in a single lane on an Illumina HiSeq 3000, with 75 base-pair paired ends.
Ruane and Austin then combined their data with UCEs from 28 snakes from a previous study, as well as UCEs from the genome of the snake Python molurus and the outgroup Anolis carolinensis.
They were able to recover quantifiable DNA from 13 samples and detected size-appropriate inserts in 10 of those (none from formalin-fixed samples and one from an alcohol-fixed sample) resulting in viable UCEs. Post-assembly, a mean of 2,318 UCEs were collected.
"The UCEs from the modern samples versus the fluid-preserved, intractable samples were comparable in most ways," the authors wrote. For example, they collected an average of 2,318 UCEs from the ancient samples compared to an average of 2,669 UCEs from the modern samples. Further, they noted an average of 24,915 parsimony informative sites across all UCEs for the intractable samples compared to an average of 31,658 parsimony informative sites for modern samples.
They did detect some differences in UCE length and an indication of higher levels of degradation in the fluid-preserved specimens. The intractable specimens had an average UCE length of 164 bp compared to an average UCE length of 468 bp for the modern samples.
Targeted capture seems ideal for these types of samples, Ruane and Austin concluded, writing, "It takes advantage of already fragmented DNA and, unlike restriction-site approaches, target capture is likely to result in replicable results across taxa, with high coverage of thousands of phylogenetically informative loci, even for low-quality samples."
The DNA they were able to extract from the ancient snakes also corroborated phylogenetic hypotheses the authors had devised from previous work.
"While this is not the first study to successfully generate DNA sequences from formalin-fixed and fluid-preserved specimens, prior work has relied mainly on time-intensive Sanger sequencing, producing only a few loci," they wrote. "In contrast, our protocol is simple, cost- and time-effective, and generates thousands of loci. We expect this will be especially useful for museum specimens that are the only samples available for certain species or specific localities."