Spatial and temporal genetic dynamics of the grasshopper Oedaleus decorus revealed by museum genomics

Analyzing genetic variation through time and space is important to identify key evolutionary and ecological processes in populations. However, using contemporary genetic data to infer the dynamics of genetic diversity may be at risk of a bias, as inferences are performed from a set of extant populations, setting aside unavailable, rare, or now extinct lineages. Here, we took advantage of new developments in next‐generation sequencing to analyze the spatial and temporal genetic dynamics of the grasshopper Oedaleus decorus, a steppic Southwestern‐Palearctic species. We applied a recently developed hybridization capture (hyRAD) protocol that allows retrieving orthologous sequences even from degraded DNA characteristic of museum specimens. We identified single nucleotide polymorphisms in 68 historical and 51 modern samples in order to (i) unravel the spatial genetic structure across part of the species distribution and (ii) assess the loss of genetic diversity over the past century in Swiss populations. Our results revealed (i) the presence of three potential glacial refugia spread across the European continent and converging spatially in the Alpine area. In addition, and despite a limited population sample size, our results indicate (ii) a loss of allelic richness in contemporary Swiss populations compared to historical populations, whereas levels of expected heterozygosities were not significantly different. This observation is compatible with an increase in the bottleneck magnitude experienced by central European populations of O. decorus following human‐mediated land‐use change impacting steppic habitats. Our results confirm that application of hyRAD to museum samples produces valuable information to study genetic processes across time and space.     

Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens

In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD) or performing size selection of the resulting fragments (in the case of single-digest RAD). Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD). In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites that usually causes loci dropout. This should enable the application of hyRAD to analyses at broader evolutionary scales.     

Sequence capture phylogenomics of historical ethanol‐preserved museum specimens: Unlocking the rest of the vault

Natural history collections play a crucial role in biodiversity research, and museum specimens are increasingly being incorporated into modern genetics‐based studies. Sequence capture methods have proven incredibly useful for phylogenomics, providing the additional ability to sequence historical museum specimens with highly degraded DNA, which until recently have been deemed less valuable for genetic work. The successful sequencing of ultraconserved elements (UCEs) from historical museum specimens has been demonstrated on multiple tissue types including dried bird skins, formalin‐fixed squamates and pinned insects. However, no study has thoroughly demonstrated this approach for historical ethanol‐preserved museum specimens. Alongside sequencing of “fresh” specimens preserved in >95% ethanol and stored at ?80°C, we used extraction techniques specifically designed for degraded DNA coupled with sequence capture protocols to sequence UCEs from historical museum specimens preserved in 70%–80% ethanol and stored at room temperature, the standard for such ethanol‐preserved museum collections. Across 35 fresh and 15 historical museum samples of the arachnid order Opiliones, an average of 345 UCE loci were included in phylogenomic matrices, with museum samples ranging from six to 495 loci. We successfully demonstrate the inclusion of historical ethanol‐preserved museum specimens in modern sequence capture phylogenomic studies, show a high frequency of variant bases at the species and population levels, and from off‐target reads successfully recover multiple loci traditionally sequenced in multilocus studies including mitochondrial loci and nuclear rRNA loci. The methods detailed in this study will allow researchers to potentially acquire genetic data from millions of ethanol‐preserved museum specimens held in collections worldwide.     

Characterizing restriction enzyme‐associated loci in historic ragweed (Ambrosia artemisiifolia) voucher specimens using custom‐designed RNA probes

Population genetic studies of nonmodel organisms frequently employ reduced representation library (RRL) methodologies, many of which rely on protocols in which genomic DNA is digested by one or more restriction enzymes. However, because high molecular weight DNA is recommended for these protocols, samples with degraded DNA are generally unsuitable for RRL methods. Given that ancient and historic specimens can provide key temporal perspectives to evolutionary questions, we explored how custom‐designed RNA probes could enrich for RRL loci (Restriction Enzyme‐Associated Loci baits, or REALbaits). Starting with genotyping‐by‐sequencing (GBS) data generated on modern common ragweed (Ambrosia artemisiifolia L.) specimens, we designed 20 000 RNA probes to target well‐characterized genomic loci in herbarium voucher specimens dating from 1835 to 1913. Compared to shotgun sequencing, we observed enrichment of the targeted loci at 19‐ to 151‐fold. Using our GBS capture pipeline on a data set of 38 herbarium samples, we discovered 22 813 SNPs, providing sufficient genomic resolution to distinguish geographic populations. For these samples, we found that dilution of REALbaits to 10% of their original concentration still yielded sufficient data for downstream analyses and that a sequencing depth of ~7m reads was sufficient to characterize most loci without wasting sequencing capacity. In addition, we observed that targeted loci had highly variable rates of success, which we primarily attribute to similarity between loci, a trait that ultimately interferes with unambiguous read mapping. Our findings can help researchers design capture experiments for RRL loci, thereby providing an efficient means to integrate samples with degraded DNA into existing RRL data sets.     

Whole mitochondrial genome capture from faecal samples and museum‐preserved specimens

Population‐scale molecular studies of endangered and cryptic species are often limited by access to high‐quality samples. The use of noninvasively collected samples or museum‐preserved specimens reduces the pressure on modern populations by removing the need to capture and handle live animals. However, endogenous DNA content in such samples is low, making shotgun sequencing a financially prohibitive approach. Here, we apply a target enrichment method to retrieve mitochondrial genomes from 65 museum specimens and 56 noninvasively collected faecal samples of two endangered great ape species, Grauer's gorilla and the eastern chimpanzee. We show that the applied method is suitable for a wide range of sample types that differ in endogenous DNA content, increasing the proportion of target reads to over 300‐fold. By systematically evaluating biases introduced during target enrichment of pooled museum samples, we show that capture is less efficient for fragments shorter or longer than the baits, that the proportion of human contaminating reads increases postcapture although capture efficiency is lower for human compared to gorilla fragments with a gorilla‐generated bait, and that the rate of jumping PCR is considerable, but can be controlled for with a double‐barcoding approach. We succeed in capturing complete mitochondrial genomes from faecal samples, but observe reduced capture efficiency as sequence divergence increases between the bait and target species. As previously shown for museum specimens, we demonstrate here that mitochondrial genome capture from field‐collected faecal samples is a robust and reliable approach for population‐wide studies of nonmodel organisms.     

Sequencing historical specimens: successful preparation of small specimens with low amounts of degraded DNA

Despite advances that allow DNA sequencing of old museum specimens, sequencing small‐bodied, historical specimens can be challenging and unreliable as many contain only small amounts of fragmented DNA. Dependable methods to sequence such specimens are especially critical if the specimens are unique. We attempt to sequence small‐bodied (3–6 mm) historical specimens (including nomenclatural types) of beetles that have been housed, dried, in museums for 58–159 years, and for which few or no suitable replacement specimens exist. To better understand ideal approaches of sample preparation and produce preparation guidelines, we compared different library preparation protocols using low amounts of input DNA (1–10 ng). We also explored low‐cost optimizations designed to improve library preparation efficiency and sequencing success of historical specimens with minimal DNA, such as enzymatic repair of DNA. We report successful sample preparation and sequencing for all historical specimens despite our low‐input DNA approach. We provide a list of guidelines related to DNA repair, bead handling, reducing adapter dimers and library amplification. We present these guidelines to facilitate more economical use of valuable DNA and enable more consistent results in projects that aim to sequence challenging, irreplaceable historical specimens.     

Sequence capture using PCR‐generated probes: a cost‐effective method of targeted high‐throughput sequencing for nonmodel organisms

Recent advances in high‐throughput sequencing library preparation and subgenomic enrichment methods have opened new avenues for population genetics and phylogenetics of nonmodel organisms. To multiplex large numbers of indexed samples while sequencing predominantly orthologous, targeted regions of the genome, we propose modifications to an existing, in‐solution capture that utilizes PCR products as target probes to enrich library pools for the genomic subset of interest. The sequence capture using PCR‐generated probes (SCPP) protocol requires no specialized equipment, is highly flexible and significantly reduces experimental costs for projects where a modest scale of genetic data is optimal (25–100 genomic loci). Our alterations enable application of this method across a wider phylogenetic range of taxa and result in higher capture efficiencies and coverage at each locus. Efficient and consistent capture over multiple SCPP experiments and at various phylogenetic distances is demonstrated, extending the utility of this method to both phylogeographic and phylogenomic studies.     

Genotyping‐in‐Thousands by sequencing (GT‐seq) panel development and application to minimally invasive DNA samples to support studies in molecular ecology

Minimally invasive sampling (MIS) is widespread in wildlife studies; however, its utility for massively parallel DNA sequencing (MPS) is limited. Poor sample quality and contamination by exogenous DNA can make MIS challenging to use with modern genotyping‐by‐sequencing approaches, which have been traditionally developed for high‐quality DNA sources. Given that MIS is often more appropriate in many contexts, there is a need to make such samples practical for harnessing MPS. Here, we test the ability for Genotyping‐in‐Thousands by sequencing (GT‐seq), a multiplex amplicon sequencing approach, to effectively genotype minimally invasive cloacal DNA samples collected from the Western Rattlesnake (Crotalus oreganus), a threatened species in British Columbia, Canada. As there was no previous genetic information for this species, an optimized panel of 362 SNPs was selected for use with GT‐seq from a de novo restriction site‐associated DNA sequencing (RADseq) assembly. Comparisons of genotypes generated within and among RADseq and GT‐seq for the same individuals found low rates of genotyping error (GT‐seq: 0.50%; RADseq: 0.80%) and discordance (2.57%), the latter likely due to the different genotype calling models employed. GT‐seq mean genotype discordance between blood and cloacal swab samples collected from the same individuals was also minimal (1.37%). Estimates of population diversity parameters were similar across GT‐seq and RADseq data sets, as were inferred patterns of population structure. Overall, GT‐seq can be effectively applied to low‐quality DNA samples, minimizing the inefficiencies presented by exogenous DNA typically found in minimally invasive samples and continuing the expansion of molecular ecology and conservation genetics in the genomics era.     

Hybridization ddRAD‐sequencing for population genomics of nonmodel plants using highly degraded historical specimen DNA

Species’ responses at the genetic level are key to understanding the long‐term consequences of anthropogenic global change. Herbaria document such responses, and, with contemporary sampling, provide high‐resolution time‐series of plant evolutionary change. Characterizing genetic diversity is straightforward for model species with small genomes and a reference sequence. For nonmodel species—with small or large genomes—diversity is traditionally assessed using restriction‐enzyme‐based sequencing. However, age‐related DNA damage and fragmentation preclude the use of this approach for ancient herbarium DNA. Here, we combine reduced‐representation sequencing and hybridization‐capture to overcome this challenge and efficiently compare contemporary and historical specimens. Specifically, we describe how homemade DNA baits can be produced from reduced‐representation libraries of fresh samples, and used to efficiently enrich historical libraries for the same fraction of the genome to produce compatible sets of sequence data from both types of material. Applying this approach to both Arabidopsis thaliana and the nonmodel plant Cardamine bulbifera, we discovered polymorphisms de novo in an unbiased, reference‐free manner. We show that the recovered genetic variation recapitulates known genetic diversity in A. thaliana, and recovers geographical origin in both species and over time, independent of bait diversity. Hence, our method enables fast, cost‐efficient, large‐scale integration of contemporary and historical specimens for assessment of genome‐wide genetic trends over time, independent of genome size and presence of a reference genome.     

Which specimens from a museum collection will yield DNA barcodes? A time series study of spiders in alcohol

We report initial results from an ongoing effort to build a library of DNA barcode sequences for Dutch spiders and investigate the utility of museum collections as a source of specimens for barcoding spiders. Source material for the library comes from a combination of specimens freshly collected in the field specifically for this project and museum specimens collected in the past. For the museum specimens, we focus on 31 species that have been frequently collected over the past several decades. A series of progressively older specimens representing these 31 species were selected for DNA barcoding. Based on the pattern of sequencing successes and failures, we find that smaller-bodied species expire before larger-bodied species as tissue sources for single-PCR standard DNA barcoding. Body size and age of oldest successful DNA barcode are significantly correlated after factoring out phylogenetic effects using independent contrasts analysis. We found some evidence that extracted DNA concentration is correlated with body size and inversely correlated with time since collection, but these relationships are neither strong nor consistent. DNA was extracted from all specimens using standard destructive techniques involving the removal and grinding of tissue. A subset of specimens was selected to evaluate nondestructive extraction. Nondestructive extractions significantly extended the DNA barcoding shelf life of museum specimens, especially small-bodied species, and yielded higher DNA concentrations compared to destructive extractions. All primary data are publically available through a Dryad archive and the Barcode of Life database.     

Utilizing field collected insects for next generation sequencing: Effects of sampling,storage, and DNA extraction methods

DNA sequencing technologies continue to advance the biological sciences, expanding opportunities for genomic studies of non‐model organisms for basic and applied questions. Despite these opportunities, many next generation sequencing protocols have been developed assuming a substantial quantity of high molecular weight DNA (>100 ng), which can be difficult to obtain for many study systems. In particular, the ability to sequence field‐collected specimens that exhibit varying levels of DNA degradation remains largely unexplored. In this study we investigate the influence of five traditional insect capture and curation methods on Double‐Digest Restriction Enzyme Associated DNA (ddRAD) sequencing success for three wild bee species. We sequenced a total of 105 specimens (between 7–13 specimens per species and treatment). We additionally investigated how different DNA quality metrics (including pre‐sequence concentration and contamination) predicted downstream sequencing success, and also compared two DNA extraction methods. We report successful library preparation for all specimens, with all treatments and extraction methods producing enough highly reliable loci for population genetic analyses. Although results varied between species, we found that specimens collected by net sampling directly into 100% EtOH, or by passive trapping followed by 100% EtOH storage before pinning tended to produce higher quality ddRAD assemblies, likely as a result of rapid specimen desiccation. Surprisingly, we found that specimens preserved in propylene glycol during field sampling exhibited lower‐quality assemblies. We provide recommendations for each treatment, extraction method, and DNA quality assessment, and further encourage researchers to consider utilizing a wider variety of specimens for genomic analyses.     

Phylogenomics using formalin‐fixed and 100+ year‐old intractable natural history specimens

Museum specimens provide a wealth of information to biologists, but obtaining genetic data from formalin‐fixed and fluid‐preserved specimens remains challenging. While DNA sequences have been recovered from such specimens, most approaches are time‐consuming and produce low data quality and quantity. Here, we use a modified DNA extraction protocol combined with high‐throughput sequencing to recover DNA from formalin‐fixed and fluid‐preserved snakes that were collected over a century ago and for which little or no modern genetic materials exist in public collections. We successfully extracted DNA and sequenced ultraconserved elements ( = 2318 loci) from 10 fluid‐preserved snakes and included them in a phylogeny with modern samples. This phylogeny demonstrates the general use of such specimens in phylogenomic studies and provides evidence for the placement of enigmatic snakes, such as the rare and never‐before sequenced Indian Xylophis stenorhynchus. Our study emphasizes the relevance of museum collections in modern research and simultaneously provides a protocol that may prove useful for specimens that have been previously intractable for DNA sequencing.     

Genomics and museum specimens

Nearly 25 years ago, Allan Wilson and colleagues isolated DNA sequences from museum specimens of kangaroo rats (Dipodomys panamintinus) and compared these sequences with those from freshly collected animals (Thomas et al. 1990 ). The museum specimens had been collected up to 78 years earlier, so the two samples provided a direct temporal comparison of patterns of genetic variation. This was not the first time DNA sequences had been isolated from preserved material, but it was the first time it had been carried out with a population sample. Population geneticists often try to make inferences about the influence of historical processes such as selection, drift, mutation and migration on patterns of genetic variation in the present. The work of Wilson and colleagues was important in part because it suggested a way in which population geneticists could actually study genetic change in natural populations through time, much the same way that experimentalists can do with artificial populations in the laboratory. Indeed, the work of Thomas et al. ( 1990 ) spawned dozens of studies in which museum specimens were used to compare historical and present‐day genetic diversity (reviewed in Wandeler et al. 2007 ). All of these studies, however, were limited by the same fundamental problem: old DNA is degraded into short fragments. As a consequence, these studies mostly involved PCR amplification of short templates, usually short stretches of mitochondrial DNA or microsatellites. In this issue, Bi et al. ( 2013 ) report a breakthrough that should open the door to studies of genomic variation in museum specimens. They used target enrichment (exon capture) and next‐generation (Illumina) sequencing to compare patterns of genetic variation in historic and present‐day population samples of alpine chipmunks (Tamias alpinus) (Fig. 1). The historic samples came from specimens collected in 1915, so the temporal span of this comparison is nearly 100 years.     

More than skin and bones: Comparing extraction methods and alternative sources of DNA from avian museum specimens

Next‐generation sequencing has greatly expanded the utility and value of museum collections by revealing specimens as genomic resources. As the field of museum genomics grows, so does the need for extraction methods that maximize DNA yields. For avian museum specimens, the established method of extracting DNA from toe pads works well for most specimens. However, for some specimens, especially those of birds that are very small or very large, toe pads can be a poor source of DNA. In this study, we apply two DNA extraction methods (phenol–chloroform and silica column) to three different sources of DNA (toe pad, skin punch and bone) from 10 historical avian museum specimens. We show that a modified phenol–chloroform protocol yielded significantly more DNA than a silica column protocol (e.g., Qiagen DNeasy Blood & Tissue Kit) across all tissue types. However, extractions using the silica column protocol contained longer fragments on average than those using the phenol–chloroform protocol, probably as a result of loss of small fragments through the silica column. While toe pads yielded more DNA than skin punches and bone fragments, skin punches proved to be a reliable alternative source of DNA and might be especially appealing when toe pad extractions are impractical. Overall, we found that historical bird museum specimens contain substantial amounts of DNA for genomic studies under most extraction scenarios, but that a phenol–chloroform protocol consistently provides the high quantities of DNA required for most current genomic protocols.     

Population genomics and comparisons of selective signatures in two invasions of melon fly, <Emphasis Type="Italic">Bactrocera cucurbitae</Emphasis> (Diptera: Tephritidae)

Population genetics is a powerful tool for invasion biology and pest management, and useful for a range of questions from tracing invasion pathways to informing management decisions with inference of population demographics. Genomics greatly increases the resolution of population-scale analyses, yet outside of model species with extensive genomic resources, few studies have used population genomics in invasion biology. We use genome-wide single nucleotide polymorphisms (SNPs) to investigate population genomic structure with samples from across the range of melon fly, Bactrocera cucurbitae (Coquillett, 1849), a highly polyphagous pest of commercial produce. We then make use of a chromosome-scale genome assembly and gene set to compare signatures of selection across the melon fly’s genome, both across our sampling as a whole and in the context of two independent, established introductions. Using multiple approaches, we find support for six genetic clusters across melon fly’s distribution. Some of these agree with previously identified genetic clusters using microsatellites, but consensus of clusters in mainland and oceanic southeast Asia is confounded by variable sampling between studies. We find few adaptive signatures across the genome, and virtually no unique signatures when comparing the two independent introductions, which suggests that similar management strategies are appropriate across melon fly’s range. This is the first use of genome-wide data to characterize population structure in tephritid fruit fly pests, and our SNP dataset provides a foundation for objective and cost-effective genotyping of previously collected melon fly specimens. Future research needs to focus on truly comprehensive sampling across melon fly’s range to overcome the historic variability of range-wide estimates of population structure for this pest.     

Museum specimens provide reliable SNP data for population genomic analysis of a widely distributed but threatened cockatoo species

Natural history museums harbour a plethora of biological specimens which are of potential use in population and conservation genetic studies. Although technical advancements in museum genomics have enabled genome‐wide markers to be generated from aged museum specimens, the suitability of these data for robust biological inference is not well characterized. The aim of this study was to test the utility of museum specimens in population and conservation genomics by assessing the biological and technical validity of single nucleotide polymorphism (SNP) data derived from such samples. To achieve this, we generated thousands of SNPs from 47 red‐tailed black cockatoo (Calyptorhychus banksii) traditional museum samples (i.e. samples that were not collected with the primary intent of DNA analysis) and 113 fresh tissue samples (cryopreserved liver/muscle) using a restriction site‐associated DNA marker approach (DArTseq^?). Thousands of SNPs were successfully generated from most of the traditional museum samples (with a mean age of 44 years, ranging from 5 to 123 years), although 38% did not provide useful data. These SNPs exhibited higher error rates and contained significantly more missing data compared with SNPs from fresh tissue samples, likely due to considerable DNA fragmentation. However, based on simulation results, the level of genotyping error had a negligible effect on inference of population structure in this species. We did identify a bias towards low diversity SNPs in older samples that appears to compromise temporal inferences of genetic diversity. This study demonstrates the utility of a RADseq‐based method to produce reliable genome‐wide SNP data from traditional museum specimens.     

Cross‐platform compatibility of de novo‐aligned SNPs in a nonmodel butterfly genus

High‐throughput sequencing methods for genotyping genome‐wide markers are being rapidly adopted for phylogenetics of nonmodel organisms in conservation and biodiversity studies. However, the reproducibility of SNP genotyping and degree of marker overlap or compatibility between datasets from different methodologies have not been tested in nonmodel systems. Using double‐digest restriction site‐associated DNA sequencing, we sequenced a common set of 22 specimens from the butterfly genus Speyeria on two different Illumina platforms, using two variations of library preparation. We then used a de novo approach to bioinformatic locus assembly and SNP discovery for subsequent phylogenetic analyses. We found a high rate of locus recovery despite differences in library preparation and sequencing platforms, as well as overall high levels of data compatibility after data processing and filtering. These results provide the first application of NGS methods for phylogenetic reconstruction in Speyeria and support the use and long‐term viability of SNP genotyping applications in nonmodel systems.     

Stacking up RADSeq assembly programs: From complete hit to completely abysmal

Decreasing sequencing costs have driven a rapid expansion of novel genotyping methods. One of these methods is the exploitation of restriction enzyme cut sites to generate genome‐wide but reduced representation sequencing libraries (RRLs), alternatively termed genotyping by sequencing or restriction‐site associated DNA sequencing. Without a reference genome, the resulting short sequence reads must be assembled de novo. There are many possible assembly programs, most not explicitly developed for RRL data, and we know little of their effectiveness. In this issue of Molecular Ecology Resources, LaCava et al. (2020) systematically evaluate six commonly used programs and two commonly varied parameters for complete and accurate assembly of RRLs, using simulated double digests of Homo sapiens and Arabidopsis thaliana genomes with varied mutation rates and types. The authors find substantial variation in performance across assembly programs. The most consistently high‐performing assembler is infrequently used in their literature survey (CD‐HIT; Li and Godzik, 2006), while several others fail to produce complete, accurate assemblies under many conditions. LaCava et al. additionally recommend best practices in parameter choice and evaluation of future assembly programs—advice that molecular ecologists working to assemble sequences of all kinds should take to heart.     

Genomic Treasure Troves: Complete Genome Sequencing of Herbarium and Insect Museum Specimens

Unlocking the vast genomic diversity stored in natural history collections would create unprecedented opportunities for genome-scale evolutionary, phylogenetic, domestication and population genomic studies. Many researchers have been discouraged from using historical specimens in molecular studies because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In today''s next-generation sequencing (NGS) world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates. Here we show that using a standard multiplex and paired-end Illumina sequencing approach, genome-scale sequence data can be generated reliably from dry-preserved plant, fungal and insect specimens collected up to 115 years ago, and with minimal destructive sampling. Using a reference-based assembly approach, we were able to produce the entire nuclear genome of a 43-year-old Arabidopsis thaliana (Brassicaceae) herbarium specimen with high and uniform sequence coverage. Nuclear genome sequences of three fungal specimens of 22–82 years of age (Agaricus bisporus, Laccaria bicolor, Pleurotus ostreatus) were generated with 81.4–97.9% exome coverage. Complete organellar genome sequences were assembled for all specimens. Using de novo assembly we retrieved between 16.2–71.0% of coding sequence regions, and hence remain somewhat cautious about prospects for de novo genome assembly from historical specimens. Non-target sequence contaminations were observed in 2 of our insect museum specimens. We anticipate that future museum genomics projects will perhaps not generate entire genome sequences in all cases (our specimens contained relatively small and low-complexity genomes), but at least generating vital comparative genomic data for testing (phylo)genetic, demographic and genetic hypotheses, that become increasingly more horizontal. Furthermore, NGS of historical DNA enables recovering crucial genetic information from old type specimens that to date have remained mostly unutilized and, thus, opens up a new frontier for taxonomic research as well.