Abstract
For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens—particularly for use in phylogenetic analyses—has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for genetic analysis.
Highlights
The primary goal of natural history museums is to preserve a biological record of the natural world for scientific study [1]
Given that the vast majority of museum specimens were collected before the advent of molecular genetics and routine collection of tissue samples, researchers have long been interested in developing protocols that would allow for the successful collection of historical DNA sequence data directly from museum specimens, even when properly prepared tissue samples were not available [2]
We report the results of these experiments and outline a minimally-destructive protocol for obtaining phylogenetically informative sequence data from formalin-fixed museum specimens
Summary
The primary goal of natural history museums is to preserve a biological record of the natural world for scientific study [1]. During approximately the past four decades, museums have become the repositories of choice for tissue samples for molecular genetic analyses, especially for non-model organisms. These tissue collections are the necessary source materials for a tremendous diversity of biological studies. Organisms prepared as study skins or dry preps, including birds, mammals and herbarium specimens are not typically exposed to formalin during preparation, and have been found to be highly amenable to hDNA data collection using traditional Sanger sequencing [3,4,5]. HDNA extraction and Sanger sequencing from museum skins of birds, mammals and ancient human remains has been used in numerous routine studies, and made possible molecular studies of extinct species [6,7,8], as well as studies of historical populations spanning both time and space [9,10,11]
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