Uncovering the genomic and metagenomic research potential in old ethanol-preserved snakes

Zacho, Claus M.; Bager, Martina Abenhus; Gravlund, Peter; Galatius, Anders; Rasmussen, Arne Redsted; Allentoft, Morten E.

doi:10.1371/journal.pone.0256353

Cited by 7 publications

(19 citation statements)

References 118 publications

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“…With older specimens, the likelihood they have been exposed to undocumented DNA-degrading conditions increases. Consistent with a study of garter snake specimens collected between 1842 and 1964 (Zacho et al, 2021), we found the age of the specimen was not strongly predictive of sequencing success, however, we did not sample specimens Both researchers and museums would benefit from an improved set of guidelines for strategic decision-making based on independent quality metrics rather than qualitative ad hoc assessments.…”

Section: Managing Expectationssupporting

confidence: 79%

Unlocking inaccessible historical genomes preserved in formalin

Hahn¹,

Alexander²,

Grealy³

et al. 2021

Molecular Ecology Resources

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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.

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Section: Managing Expectationssupporting

confidence: 79%

Unlocking inaccessible historical genomes preserved in formalin

Hahn¹,

Alexander²,

Grealy³

et al. 2021

Molecular Ecology Resources

View full text Add to dashboard Cite

show abstract

“…If available, a variety of tissue types (liver, muscle, and bone) from multiple individuals of different ages, should be used. Some studies have found that higher DNA yields were extracted from soft tissues, such as liver and muscle, compared to hard bone tissue (Zacho et al, 2021). Though, bone tissue can yield surprisingly high amounts of DNA (Zacho et al, 2021) of sufficient quality for sequencing, as we found from our homalopsid specimens, potentially due to greater protection against chemicals inside dense tissue.…”

Section: Dna Extractionmentioning

confidence: 49%

“…Some studies have found that higher DNA yields were extracted from soft tissues, such as liver and muscle, compared to hard bone tissue (Zacho et al, 2021). Though, bone tissue can yield surprisingly high amounts of DNA (Zacho et al, 2021) of sufficient quality for sequencing, as we found from our homalopsid specimens, potentially due to greater protection against chemicals inside dense tissue. Specifically, the two bone samples used here, with few nuclear loci, provided ∼66 and ∼89% of the mitochondrial genome.…”

Section: Dna Extractionmentioning

confidence: 49%

“…Hot alkali treatments (Campos and Gilbert, 2012;Hykin et al, 2015), heavy use of proteinase-K (Ruane and Austin, 2017), and development of digestion buffers (Allentoft et al, 2015) have all been used with varying success to break formalin cross-links and retrieve DNA from fixed specimens. However, different tissues (e.g., skin, liver, muscle, and bone) may yield varying DNA concentrations upon extraction (Appleyard et al, 2021;Zacho et al, 2021), and the lysis of soft tissue using enzymes like proteinase-K may be unsuccessful depending on the age, storage conditions, and preservation history of the source tissue. Even if DNA is extracted from intractable specimens, decreases in number and uneven distribution of mapped reads to reference genomes (Hykin et al, 2015;Allentoft et al, 2018), short fragment lengths, and low numbers of loci (Ruane and Austin, 2017) are commonly reported.…”

Section: Introductionmentioning

confidence: 99%

“…Snakes are an excellent system for studying evolutionary processes (Esquerré et al, 2020;Schield et al, 2020;Westeen et al, 2020;Burbrink et al, 2021), and the utilization of preserved museum specimens has expanded our knowledge on both extant and extinct diversity (Ruane and Austin, 2017;Allentoft et al, 2018;Zacho et al, 2021). Southeast Asia in particular includes a diverse assemblage of snakes with multiple endemic lineages, many concentrated in biodiversity hotspots, which have been affected by the region's complex geological history (Hall, 2009;de Bruyn et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

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Maximizing Molecular Data From Low-Quality Fluid-Preserved Specimens in Natural History Collections

Bernstein

Ruane

2022

Front. Ecol. Evol.

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Over the past decade, museum genomics studies have focused on obtaining DNA of sufficient quality and quantity for sequencing from fluid-preserved natural history specimens, primarily to be used in systematic studies. While these studies have opened windows to evolutionary and biodiversity knowledge of many species worldwide, published works often focus on the success of these DNA sequencing efforts, which is undoubtedly less common than obtaining minimal or sometimes no DNA or unusable sequence data from specimens in natural history collections. Here, we attempt to obtain and sequence DNA extracts from 115 fresh and 41 degraded samples of homalopsid snakes, as well as from two degraded samples of a poorly known snake, Hydrablabes periops. Hydrablabes has been suggested to belong to at least two different families (Natricidae and Homalopsidae) and with no fresh tissues known to be available, intractable museum specimens currently provide the only opportunity to determine this snake’s taxonomic affinity. Although our aim was to generate a target-capture dataset for these samples, to be included in a broader phylogenetic study, results were less than ideal due to large amounts of missing data, especially using the same downstream methods as with standard, high-quality samples. However, rather than discount results entirely, we used mapping methods with references and pseudoreferences, along with phylogenetic analyses, to maximize any usable molecular data from our sequencing efforts, identify the taxonomic affinity of H. periops, and compare sequencing success between fresh and degraded tissue samples. This resulted in largely complete mitochondrial genomes for five specimens and hundreds to thousands of nuclear loci (ultra-conserved loci, anchored-hybrid enrichment loci, and a variety of loci frequently used in squamate phylogenetic studies) from fluid-preserved snakes, including a specimen of H. periops from the Field Museum of Natural History collection. We combined our H. periops data with previously published genomic and Sanger-sequenced datasets to confirm the familial designation of this taxon, reject previous taxonomic hypotheses, and make biogeographic inferences for Hydrablabes. A second H. periops specimen, despite being seemingly similar for initial raw sequencing results and after being put through the same protocols, resulted in little usable molecular data. We discuss the successes and failures of using different pipelines and methods to maximize the products from these data and provide expectations for others who are looking to use DNA sequencing efforts on specimens that likely have degraded DNA.Life Science Identifier (Hydrablabes periops)urn:lsid:zoobank.org:pub:F2AA44 E2-D2EF-4747-972A-652C34C2C09D.

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Library preparation method and DNA source influence endogenous DNA recovery from 100‐year‐old avian museum specimens

Settlecowski

Marks

Manthey

2023

Ecology and Evolution

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Museum specimens collected prior to cryogenic tissue storage are increasingly being used as genetic resources, and though high‐throughput sequencing is becoming more cost‐efficient, whole genome sequencing (WGS) of historical DNA (hDNA) remains inefficient and costly due to its short fragment sizes and high loads of exogenous DNA, among other factors. It is also unclear how sequencing efficiency is influenced by DNA sources. We aimed to identify the most efficient method and DNA source for collecting WGS data from avian museum specimens. We analyzed low‐coverage WGS from 60 DNA libraries prepared from four American Robin (Turdus migratorius) and four Abyssinian Thrush (Turdus abyssinicus) specimens collected in the 1920s. We compared DNA source (toepad versus incision‐line skin clip) and three library preparation methods: (1) double‐stranded DNA (dsDNA), single tube (KAPA); (2) single‐stranded DNA (ssDNA), multi‐tube (IDT); and (3) ssDNA, single tube (Claret Bioscience). We found that the ssDNA, multi‐tube method resulted in significantly greater endogenous DNA content, average read length, and sequencing efficiency than the other tested methods. We also tested whether a predigestion step reduced exogenous DNA in libraries from one specimen per species and found promising results that warrant further study. The ~10% increase in average sequencing efficiency of the best‐performing method over a commonly implemented dsDNA library preparation method has the potential to significantly increase WGS coverage of hDNA from bird specimens. Future work should evaluate the threshold for specimen age at which these results hold and how the combination of library preparation method and DNA source influence WGS in other taxa.

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Uncovering the genomic and metagenomic research potential in old ethanol-preserved snakes

Cited by 7 publications

References 118 publications

Unlocking inaccessible historical genomes preserved in formalin

Unlocking inaccessible historical genomes preserved in formalin

Maximizing Molecular Data From Low-Quality Fluid-Preserved Specimens in Natural History Collections

Library preparation method and DNA source influence endogenous DNA recovery from 100‐year‐old avian museum specimens

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