Assessing DNA for fish identifications from reference collections: the good, bad and ugly shed light on formalin fixation and sequencing approaches

Abstract

Natural history collections are repositories of biodiversity and are potentially used by molecular ecologists for comparative taxonomic, phylogenetic, biogeographic and forensic purposes. Specimens in fish collections are preserved using a combination of methods with many fixed in formalin and then preserved in ethanol for long-term storage. Formalin fixation damages DNA, thereby limiting genetic analyses. In this study, the authors compared the DNA barcoding and identification success for frozen and formalin-fixed tissues obtained from specimens in the CSIRO Australian National Fish Collection. They studied 230 samples from fishes (consisting of >160 fish species). An optimized formalin-fixed, paraffin-embedded DNA extraction method resulted in usable DNA from degraded tissues. Four mini barcoding assays of the mitochondrial DNA (mtDNA) were characterized with Sanger and Illumina amplicon sequencing. In the good quality DNA (without exposure to formalin), up to 88% of the specimens were correctly matched at the species level using the cytochrome oxidase subunit 1 (COI) mini barcodes, whereas up to 58% of the specimens exposed to formalin for less than 8 weeks were correctly identified to species. In contrast, 16S primers provided higher amplification success with formalin-exposed tissues, although the COI gene was more successful for identification. Importantly, the authors found that DNA of a certain size and quality can be amplified and sequenced despite exposure to formalin, and Illumina sequencing provided them with greater power of resolution for taxa identification even when there was little DNA present.Overall, within parameter constraints, this study highlights the possibilities of recovering DNA barcodes for identification from formalin-fixed fish specimens, and the authors provide guidelines for when successful identification could be expected.

Unlocking inaccessible historical genomes preserved in formalin

“…Genomic study of formalin-preserved museum specimens has lagged behind because DNA extracted from such tissues is typically low-yield and highly fragmented. PCR amplification of formalin-degraded DNA templates is generally restricted to few, short genomic loci, which provide limited phylogenetic resolution (20). Formalin fixation presents further challenges by inducing numerous molecular lesions, such as strand breaks, base misincorporation, and both intra-and intermolecular cross-links (21)(22)(23).…”

“…Formaldehyde damage to DNA templates can result in sequencing artefacts that are difficult to differentiate from true genetic variants (22,23). Because PCR amplification of damaged DNA is particularly prone to sequencing artefacts, it is preferable to perform deep next-generation sequencing of amplicons (20) or to avoid amplicon approaches altogether through whole genome sequencing (WGS) of degraded templates (24). Coupled with library preparation methods optimized for low-input and damaged DNA templates (25,26), high-throughput sequencing can generate enough coverage to call genomic variants with high confidence (27).…”

“…Indeed, many specimens can be in contact with formaldehyde (or its derivatives, such as formic acid) for the entirety of their tenure in a collection. Prolonged formaldehyde exposure, especially under acidic conditions, is thought to result in more extreme DNA degradation (20,31). The damage resulting from the preservation process compounds with DNA damage due to natural decomposition, which can be extensive and often precedes any obvious visual indicators of decomposition (32).…”

“…Of the few genetic studies of formalin-fixed museum specimens, most have targeted nuclear (33)(34)(35)(36)(37) and high copy mitochondrial (20,38,39) loci via PCR amplification due to the difficulty and unpredictability of nuclear DNA extraction. There are few examples of broaderscale genomic sequencing of formalin-fixed museum specimens and none have recovered whole vertebrate genomes.…”

“…Hybridization capture baits have also been used to recover the mitochondrial genome from a 120-year-old formalin-preserved Crimean green lizard (43). Highlighting the difficulty of recovering gDNA from formalin-preserved specimens, numerous studies have reported failure to extract and amplify gDNA from formalin-preserved museum tissues (20,44,45). In this context, it is unfortunate yet wise to be hesitant to conduct destructive sampling of formalinpreserved specimens for the purposes of costly WGS.…”