Unlocking inaccessible historical genomes preserved in formalin

Abstract

Museum specimens represent an unparalleled record of historical genomic data.However, the widespread practice of formalin preservation has thus far impeded genomic analysis of a large proportion of specimens. Limited DNA sequencing from formalin-preserved specimens has yielded low genomic coverage with unpredictable success. We set out to refine sample processing methods and to identify specimen characteristics predictive of sequencing success. With a set of taxonomically diverse specimens collected between 1962 and 2006 and ranging in preservation quality, we compared the efficacy of several end-to-end whole genome sequencing workflows alongside a k-mer-based trimming-free read alignment approach to maximize mapping of endogenous sequence. We recovered complete mitochondrial genomes and up to 3× nuclear genome coverage from formalin-preserved tissues. Hot alkaline lysis coupled with phenol-chloroform extraction out-performed proteinase K digestion in recovering DNA, while library preparation method had little impact on sequencing success. The strongest predictor of DNA yield was overall specimen condition, which additively interacts with preservation conditions to accelerate DNA degradation. Here, we demonstrate a significant advance in capability beyond limited recovery of a small number of loci via PCR or target-capture sequencing. To facilitate strategic selection of suitable specimens for genomic sequencing, we present a decision-making framework that utilizes independent and nondestructive assessment criteria. Sequencing of formalin-preserved specimens will contribute to a greater understanding of temporal trends in genetic adaptation, including those associated with a changing climate. Our work enhances the value of museum collections worldwide by unlocking genomes of specimens that have been disregarded as a valid molecular resource.

Taxonomic investigation of the zooplanktivorous Lake Malawi cichlids Copadichromis mloto (Iles) and C. virginalis (Iles)

“…Notably, none of the H. mloto type material shows any sign of male breeding colours. Final certainty about the genetic identity of the type specimens relative to recent samples could only be obtained from their sequencing, which would be challenging given their preservation in formalin, but might become possible in the future (e.g., see Hahn et al, 2021). For the time being, the results presented here support the original assessment by Eccles & Trewavas (1989) that most pure utaka specimens commonly found in trawl catches correspond to C. mloto as do the species illustrated on Konings 1990 p. 256 (middle photo), Turner 1996 p. 53 and discussed on p. 67-69 as C. virginalis.…”

Taxonomic Identification of Two Poorly Known Lantern Shark Species Based on Mitochondrial DNA From Wet-Collection Paratypes

“…To make matters worse, a substantial fraction of lantern shark diversity is known only from formalin preserved type material that was collected prior to the advent of DNA sequencing. Hence, tissue sampling, common practice today for performing DNA sequence-based analysis such as DNA barcoding (Hebert et al, 2003), was not conducted and fixation in formaldehyde and preservation in ethanol causes DNA damage (Gilbert et al, 2007;Hoffman et al, 2015;Hykin et al, 2015;Stiller et al, 2016;McGuire et al, 2018;Hahn et al, 2021). This means that, while we know that the group has diversified extensively (e.g., Straube et al, 2011a;Ebert et al, 2016Ebert et al, , 2021White et al, 2017;Dolganov and Balanov, 2018), it has been hard to decipher how the different species are related to one another and how different ecological pressures have contributed to their diversification.…”

“…This means that, while we know that the group has diversified extensively (e.g., Straube et al, 2011a;Ebert et al, 2016Ebert et al, , 2021White et al, 2017;Dolganov and Balanov, 2018), it has been hard to decipher how the different species are related to one another and how different ecological pressures have contributed to their diversification. Recently developed tools allow us to obtain DNA sequence data from formalin preserved animals (Gansauge et al, 2017;Hahn et al, 2021;Straube et al, 2021a). In the current contribution we have applied these tools to type material for two species of Etmopterus and show how the data collected have implications, not only for understanding their taxonomy and evolution, but also their ranges, which has consequences for their conservation and management.…”

Ancient DNA from the extinct New Zealand grayling (Prototroctes oxyrhynchus) reveals evidence for Miocene marine dispersal

“…changes to preservative fluids, most commonly unbuffered formalin to ethanol). Until recently, obtaining ancient DNA from formalin-fixed specimens has been challenging, as fixation in formalin rapidly degrades DNA (through fragmentation and base pair modification) and promotes cross-linkage both within and between DNA molecules, as well as to cellular proteins; both of which accumulate linearly with increased preservation time (Hykin et al, 2015; Hahn et al, 2021). However, methodological and technological advancements—especially the advent of high-throughput DNA sequencing—have dramatically improved success rates of obtaining genetic data from such fluid-preserved specimens.…”