Squeezing water from a stone: high-throughput sequencing from a 145-year old holotype resolves (barely) a cryptic species problem in flying lizards

We used Massively Parallel High-Throughput Sequencing to obtain genetic data from a 145-year old holotype specimen of the flying lizard, Draco cristatellus. Obtaining genetic data from this holotype was necessary to resolve an otherwise intractable taxonomic problem involving the status of this species relative to closely related sympatric Draco species that cannot otherwise be distinguished from one another on the basis of museum specimens. Initial analyses suggested that the DNA present in the holotype sample was so degraded as to be unusable for sequencing. However, we used a specialized extraction procedure developed for highly degraded ancient DNA samples and MiSeq shotgun sequencing to obtain just enough low-coverage mitochondrial DNA (721 base pairs) to conclusively resolve the species status of the holotype as well as a second known specimen of this species. The holotype was prepared before the advent of formalin-fixation and therefore was most likely originally fixed with ethanol and never exposed to formalin. Whereas conventional wisdom suggests that formalin-fixed samples should be the most challenging for DNA sequencing, we propose that evaporation during long-term alcohol storage and consequent water-exposure may subject older ethanol-fixed museum specimens to hydrolytic damage. If so, this may pose an even greater challenge for sequencing efforts involving historical samples.

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Squeezing water from a stone: High-throughput sequencing from a 145-year old holotype resolves (barely) a cryptic species problem in flying lizards

10.7287/peerj.preprints.26466 ◽

2018 ◽

Cited By ~ 1

Author(s):

Jimmy A McGuire ◽

Darko D Cotoras ◽

Brendan O'Connell ◽

Shobi Z S Lawalata ◽

Cynthia Y Wang-Claypool ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Extraction Procedure ◽

Genetic Data ◽

Museum Specimens ◽

Base Pairs ◽

Holotype Specimen ◽

The Status ◽

Taxonomic Problem ◽

Low Coverage

We used Massively Parallel High-Throughput Sequencing to obtain genetic data from a 145-year old holotype specimen of the flying lizard, Draco cristatellus. Obtaining genetic data from this holotype was necessary to resolve an otherwise intractable taxonomic problem involving the status of this species relative to closely related sympatric Draco species that cannot otherwise be distinguished from one another on the basis of museum specimens. Initial analyses suggested that the DNA present in the holotype sample was so degraded as to be unusable for sequencing. However, we used a specialized extraction procedure developed for highly degraded ancient DNA samples and MiSeq shotgun sequencing to obtain just enough low-coverage mitochondrial DNA (547 base pairs) to conclusively resolve the species status of the holotype as well as a second known specimen of this species. The holotype was prepared before the advent of formalin-fixation and therefore was most likely originally fixed with ethanol and never exposed to formalin. Whereas conventional wisdom suggests that formalin-fixed samples should be the most challenging for DNA sequencing, we propose that evaporation during long-term alcohol storage and consequent water-exposure may subject older ethanol-fixed museum specimens to hydrolytic damage. If so, this may pose an even greater challenge for sequencing efforts involving historical samples.

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Squeezing water from a stone: High-throughput sequencing from a 145-year old holotype resolves (barely) a cryptic species problem in flying lizards

10.7287/peerj.preprints.26466v1 ◽

2018 ◽

Cited By ~ 1

Author(s):

Jimmy A McGuire ◽

Darko D Cotoras ◽

Brendan O'Connell ◽

Shobi Z S Lawalata ◽

Cynthia Y Wang-Claypool ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Extraction Procedure ◽

Genetic Data ◽

Museum Specimens ◽

Base Pairs ◽

Holotype Specimen ◽

The Status ◽

Taxonomic Problem ◽

Low Coverage

We used Massively Parallel High-Throughput Sequencing to obtain genetic data from a 145-year old holotype specimen of the flying lizard, Draco cristatellus. Obtaining genetic data from this holotype was necessary to resolve an otherwise intractable taxonomic problem involving the status of this species relative to closely related sympatric Draco species that cannot otherwise be distinguished from one another on the basis of museum specimens. Initial analyses suggested that the DNA present in the holotype sample was so degraded as to be unusable for sequencing. However, we used a specialized extraction procedure developed for highly degraded ancient DNA samples and MiSeq shotgun sequencing to obtain just enough low-coverage mitochondrial DNA (547 base pairs) to conclusively resolve the species status of the holotype as well as a second known specimen of this species. The holotype was prepared before the advent of formalin-fixation and therefore was most likely originally fixed with ethanol and never exposed to formalin. Whereas conventional wisdom suggests that formalin-fixed samples should be the most challenging for DNA sequencing, we propose that evaporation during long-term alcohol storage and consequent water-exposure may subject older ethanol-fixed museum specimens to hydrolytic damage. If so, this may pose an even greater challenge for sequencing efforts involving historical samples.

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Assessing genotyping errors in mammalian museum study skins using high-throughput genotyping-by-sequencing

Conservation Genetics Resources ◽

10.1007/s12686-021-01213-8 ◽

2021 ◽

Author(s):

Stella C. Yuan ◽

Eric Malekos ◽

Melissa T. R. Hawkins

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Massively Parallel Sequencing ◽

Massively Parallel ◽

Museum Specimens ◽

Museum Specimen ◽

Genotyping Errors ◽

Allelic Dropout ◽

Parallel Sequencing ◽

Sequencing Technologies

AbstractThe use of museum specimens held in natural history repositories for population and conservation genetic research is increasing in tandem with the use of massively parallel sequencing technologies. Short Tandem Repeats (STRs), or microsatellite loci, are commonly used genetic markers in wildlife and population genetic studies. However, they traditionally suffered from a host of issues including length homoplasy, high costs, low throughput, and difficulties in reproducibility across laboratories. Massively parallel sequencing technologies can address these problems, but the incorporation of museum specimen derived DNA suffers from significant fragmentation and exogenous DNA contamination. Combatting these issues requires extra measures of stringency in the lab and during data analysis, yet there have not been any high-throughput sequencing studies evaluating microsatellite allelic dropout from museum specimen extracted DNA. In this study, we evaluate genotyping errors derived from mammalian museum skin DNA extracts for previously characterized microsatellites across PCR replicates utilizing high-throughput sequencing. We found it useful to classify samples based on DNA concentration, which determined the rate by which genotypes were accurately recovered. Longer microsatellites performed worse in all museum specimens. Allelic dropout rates across loci were dependent on sample quantity, with high concentration museum specimens performing as well and recovering quality metrics nearly as high as the frozen tissue sample. Based on our results, we provide a set of best practices for quality assurance and incorporation of reliable genotypes from museum specimens.

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11 Million SNP Reference Profiles for Identity Searching Across Ethnicities, Kinship, and Admixture

10.1101/321190 ◽

2018 ◽

Author(s):

Brian S. Helfer ◽

Darrell O. Ricke

Keyword(s):

Single Nucleotide Polymorphisms ◽

High Throughput ◽

Family Relationships ◽

In Silico ◽

High Throughput Sequencing ◽

Genetic Data ◽

Nucleotide Polymorphisms ◽

Mixture Analysis ◽

Single Nucleotide ◽

Public Repositories

AbstractHigh throughput sequencing (HTS) of single nucleotide polymorphisms (SNPs) provides additional applications for DNA forensics including identification, mixture analysis, kinship prediction, and biogeographic ancestry prediction. Public repositories of human genetic data are being rapidly generated and released, but the majorities of these samples are de-identified to protect privacy, and have little or no individual metadata such as appearance (photos), ethnicity, relatives, etc. A reference in silico dataset has been generated to enable development and testing of new DNA forensics algorithms. This dataset provides 11 million SNP profiles for individuals with defined ethnicities and family relationships spanning eight generations with admixture for a panel with 39,108 SNPs.

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sppIDer: a species identification tool to investigate hybrid genomes with high-throughput sequencing

10.1101/333815 ◽

2018 ◽

Cited By ~ 1

Author(s):

Quinn K. Langdon ◽

David Peris ◽

Brian Kyle ◽

Chris Todd Hittinger

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Rapid Identification ◽

Sequencing Data ◽

Pure Species ◽

High Throughput Sequencing Data ◽

Interspecies Hybrids ◽

Evolutionary Trajectories ◽

Low Coverage ◽

Reference Genomes

AbstractThe genomics era has expanded our knowledge about the diversity of the living world, yet harnessing high-throughput sequencing data to investigate alternative evolutionary trajectories, such as hybridization, is still challenging. Here we present sppIDer, a pipeline for the characterization of interspecies hybrids and pure species,that illuminates the complete composition of genomes. sppIDer maps short-read sequencing data to a combination genome built from reference genomes of several species of interest and assesses the genomic contribution and relative ploidy of each parental species, producing a series of colorful graphical outputs ready for publication. As a proof-of-concept, we use the genus Saccharomyces to detect and visualize both interspecies hybrids and pure strains, even with missing parental reference genomes. Through simulation, we show that sppIDer is robust to variable reference genome qualities and performs well with low-coverage data. We further demonstrate the power of this approach in plants, animals, and other fungi. sppIDer is robust to many different inputs and provides visually intuitive insight into genome composition that enables the rapid identification of species and their interspecies hybrids. sppIDer exists as a Docker image, which is a reusable, reproducible, transparent, and simple-to-run package that automates the pipeline and installation of the required dependencies (https://github.com/GLBRC/sppIDer).

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Understanding “Alexandrian” extinctions using genomic DNA from fluid-preserved museum specimens of Desmognathus salamanders

10.21203/rs.3.rs-769753/v1 ◽

2021 ◽

Author(s):

Alex Pyron ◽

David A. Beamer ◽

Chace R. Holzheuser ◽

Emily Moriarty Lemmon ◽

Alan R. Lemmon ◽

...

Keyword(s):

Natural History ◽

Large Scale ◽

Extraction Procedure ◽

Genetic Data ◽

Museum Specimens ◽

Population Declines ◽

Sandy Bottom ◽

Sequencing Errors ◽

Plethodontid Salamander ◽

Anchored Hybrid Enrichment

Abstract Species that went extinct prior to the genomic era are typically out-of-reach for modern phylogenetic studies. We refer to these as “Alexandrian” extinctions, after the lost library of the ancient world. This is particularly limiting for conservation studies, as genetic data for such taxa may be key to understand extinction threats and risks, the causes of declines, and inform management of related, extant populations. Fortunately, continual advances in biochemistry and DNA sequencing offer increasing ability to recover DNA from historical museum specimens, including fluid-preserved natural history collections. Here, we report on success in recovering nuclear and mitochondrial data from the apparently-extinct subspecies Desmognathus fuscus carri (Neill 1951), a plethodontid salamander from spring runs in central Florida. The two specimens are 50 years old and were likely preserved in unbuffered formalin, but application of a recently derived extraction procedure yielded usable DNA and partially successful Anchored Hybrid Enrichment sequencing. These data suggest that the populations of D. f. carri from peninsular Florida are conspecific with the D. auriculatus A lineage as suggested by previous authors, but likely represented an ecogeographically distinct genetic segment that has now been lost. Genetic data from this Alexandrian extinction thus confirm the geographic extent of population declines and extirpations as well as their ecological context, suggesting a possibly disproportionate loss from sandy-bottom clearwater streams compared to blackwater swamps. Success of these methods bodes well for large-scale application to fluid-preserved natural history specimens from relevant historical populations, but the possibility of significant DNA damage and related sequencing errors in additional hurdle to overcome.

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High-Throughput Sequencing of 16S rRNA Gene Amplicons: Effects of Extraction Procedure, Primer Length and Annealing Temperature

PLoS ONE ◽

10.1371/journal.pone.0038094 ◽

2012 ◽

Vol 7 (5) ◽

pp. e38094 ◽

Cited By ~ 52

Author(s):

Martin J. Sergeant ◽

Chrystala Constantinidou ◽

Tristan Cogan ◽

Charles W. Penn ◽

Mark J. Pallen

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

High Throughput ◽

High Throughput Sequencing ◽

Annealing Temperature ◽

Extraction Procedure ◽

Rrna Gene

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Accounting for Errors in Low Coverage High-Throughput Sequencing Data when Constructing Genetic Maps using Biparental Outcrossed Populations

10.1101/249722 ◽

2018 ◽

Author(s):

Timothy P. Bilton ◽

Matthew R. Schofield ◽

Michael A. Black ◽

David Chagné ◽

Phillip L. Wilcox ◽

...

Keyword(s):

High Throughput ◽

Genetic Linkage ◽

High Throughput Sequencing ◽

Diploid Species ◽

Genotyping By Sequencing ◽

Genetic Maps ◽

Linkage Maps ◽

Sequencing Data ◽

Genetic Linkage Maps ◽

Low Coverage

ABSTRACTNext generation sequencing is an efficient method that allows for substantially more markers than previous technologies, providing opportunities for building high density genetic linkage maps, which facilitate the development of non-model species’ genomic assemblies and the investigation of their genes. However, constructing genetic maps using data generated via high-throughput sequencing technology (e.g., genotyping-by-sequencing) is complicated by the presence of sequencing errors and genotyping errors resulting from missing parental alleles due to low sequencing depth. If unaccounted for, these errors lead to inflated genetic maps. In addition, map construction in many species is performed using full-sib family populations derived from the outcrossing of two individuals, where unknown parental phase and varying segregation types further complicate construction. We present a new methodology for modeling low coverage sequencing data in the construction of genetic linkage maps using full-sib populations of diploid species, implemented in a package called GUSMap. Our model is based on an extension of the Lander-Green hidden Markov model that accounts for errors present in sequencing data. Results show that GUSMap was able to give accurate estimates of the recombination fractions and overall map distance, while most existing mapping packages produced inflated genetic maps in the presence of errors. Our results demonstrate the feasibility of using low coverage sequencing data to produce genetic maps without requiring extensive filtering of potentially erroneous genotypes, provided that the associated errors are correctly accounted for in the model.

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