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.     

Unlocking the secondary gene‐pool of barley with next‐generation sequencing

Crop wild relatives (CWR) provide an important source of allelic diversity for any given crop plant species for counteracting the erosion of genetic diversity caused by domestication and elite breeding bottlenecks. Hordeum bulbosum L. is representing the secondary gene pool of the genus Hordeum. It has been used as a source of genetic introgressions for improving elite barley germplasm (Hordeum vulgare L.). However, genetic introgressions from H. bulbosum have yet not been broadly applied, due to a lack of suitable molecular tools for locating, characterizing, and decreasing by recombination and marker‐assisted backcrossing the size of introgressed segments. We applied next‐generation sequencing (NGS) based strategies for unlocking genetic diversity of three diploid introgression lines of cultivated barley containing chromosomal segments of its close relative H. bulbosum. Firstly, exome capture‐based (re)‐sequencing revealed large numbers of single nucleotide polymorphisms (SNPs) enabling the precise allocation of H. bulbosum introgressions. This SNP resource was further exploited by designing a custom multiplex SNP genotyping assay. Secondly, two‐enzyme‐based genotyping‐by‐sequencing (GBS) was employed to allocate the introgressed H. bulbosum segments and to genotype a mapping population. Both methods provided fast and reliable detection and mapping of the introgressed segments and enabled the identification of recombinant plants. Thus, the utilization of H. bulbosum as a resource of natural genetic diversity in barley crop improvement will be greatly facilitated by these tools in the future.     

Museum epigenomics: Characterizing cytosine methylation in historic museum specimens

Museum genomics has transformed the field of collections‐based research, opening up a range of new research directions for paleontological specimens as well as natural history specimens collected over the past few centuries. Recent work demonstrates that it is possible to characterize epigenetic markers such as DNA methylation in well preserved ancient tissues. This approach has not yet been tested in traditionally prepared natural history specimens such as dried bones and skins, the most common specimen types in vertebrate collections. In this study, we developed and tested methods to characterize cytosine methylation in dried skulls up to 76 years old. Using a combination of ddRAD and bisulphite treatment, we characterized patterns of cytosine methylation in two species of deer mouse (Peromyscus spp.) collected in the same region in Michigan in 1940, 2003, and 2013–2016. We successfully estimated methylation in specimens of all age groups, although older specimens yielded less data and showed greater interindividual variation in data yield than newer specimens. Global methylation estimates were reduced in the oldest specimens (76 years old) relative to the newest specimens (1–3 years old), which may reflect post‐mortem hydrolytic deamination. Methylation was reduced in promoter regions relative to gene bodies and showed greater bimodality in autosomes relative to female X chromosomes, consistent with expectations for methylation in mammalian somatic cells. Our work demonstrates the utility of historic specimens for methylation analyses, as with genomic analyses; however, studies will need to accommodate the large variance in the quantity of data produced by older specimens.     

exonsampler: a computer program for genome‐wide and candidate gene exon sampling for targeted next‐generation sequencing

The computer program exonsampler automates the sampling of thousands of exon sequences from publicly available reference genome sequences and gene annotation databases. It was designed to provide exon sequences for the efficient, next‐generation gene sequencing method called exon capture. The exon sequences can be sampled by a list of gene name abbreviations (e.g. IFNG, TLR1), or by sampling exons from genes spaced evenly across chromosomes. It provides a list of genomic coordinates (a bed file), as well as a set of sequences in fasta format. User‐adjustable parameters for collecting exon sequences include a minimum and maximum acceptable exon length, maximum number of exonic base pairs (bp) to sample per gene, and maximum total bp for the entire collection. It allows for partial sampling of very large exons. It can preferentially sample upstream (5 prime) exons, downstream (3 prime) exons, both external exons, or all internal exons. It is written in the Python programming language using its free libraries. We describe the use of exonsampler to collect exon sequences from the domestic cow (Bos taurus) genome for the design of an exon‐capture microarray to sequence exons from related species, including the zebu cow and wild bison. We collected ~10% of the exome (~3 million bp), including 155 candidate genes, and ~16 000 exons evenly spaced genomewide. We prioritized the collection of 5 prime exons to facilitate discovery and genotyping of SNPs near upstream gene regulatory DNA sequences, which control gene expression and are often under natural selection.     

Targeted multiplex next‐generation sequencing: advances in techniques of mitochondrial and nuclear DNA sequencing for population genomics

Next‐generation sequencing (NGS) is emerging as an efficient and cost‐effective tool in population genomic analyses of nonmodel organisms, allowing simultaneous resequencing of many regions of multi‐genomic DNA from multiplexed samples. Here, we detail our synthesis of protocols for targeted resequencing of mitochondrial and nuclear loci by generating indexed genomic libraries for multiplexing up to 100 individuals in a single sequencing pool, and then enriching the pooled library using custom DNA capture arrays. Our use of DNA sequence from one species to capture and enrich the sequencing libraries of another species (i.e. cross‐species DNA capture) indicates that efficient enrichment occurs when sequences are up to about 12% divergent, allowing us to take advantage of genomic information in one species to sequence orthologous regions in related species. In addition to a complete mitochondrial genome on each array, we have included between 43 and 118 nuclear loci for low‐coverage sequencing of between 18 kb and 87 kb of DNA sequence per individual for single nucleotide polymorphisms discovery from 50 to 100 individuals in a single sequencing lane. Using this method, we have generated a total of over 500 whole mitochondrial genomes from seven cetacean species and green sea turtles. The greater variation detected in mitogenomes relative to short mtDNA sequences is helping to resolve genetic structure ranging from geographic to species‐level differences. These NGS and analysis techniques have allowed for simultaneous population genomic studies of mtDNA and nDNA with greater genomic coverage and phylogeographic resolution than has previously been possible in marine mammals and turtles.     

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.     

Next‐generation DNA barcoding: using next‐generation sequencing to enhance and accelerate DNA barcode capture from single specimens

DNA barcoding is an efficient method to identify specimens and to detect undescribed/cryptic species. Sanger sequencing of individual specimens is the standard approach in generating large‐scale DNA barcode libraries and identifying unknowns. However, the Sanger sequencing technology is, in some respects, inferior to next‐generation sequencers, which are capable of producing millions of sequence reads simultaneously. Additionally, direct Sanger sequencing of DNA barcode amplicons, as practiced in most DNA barcoding procedures, is hampered by the need for relatively high‐target amplicon yield, coamplification of nuclear mitochondrial pseudogenes, confusion with sequences from intracellular endosymbiotic bacteria (e.g. Wolbachia) and instances of intraindividual variability (i.e. heteroplasmy). Any of these situations can lead to failed Sanger sequencing attempts or ambiguity of the generated DNA barcodes. Here, we demonstrate the potential application of next‐generation sequencing platforms for parallel acquisition of DNA barcode sequences from hundreds of specimens simultaneously. To facilitate retrieval of sequences obtained from individual specimens, we tag individual specimens during PCR amplification using unique 10‐mer oligonucleotides attached to DNA barcoding PCR primers. We employ 454 pyrosequencing to recover full‐length DNA barcodes of 190 specimens using 12.5% capacity of a 454 sequencing run (i.e. two lanes of a 16 lane run). We obtained an average of 143 sequence reads for each individual specimen. The sequences produced are full‐length DNA barcodes for all but one of the included specimens. In a subset of samples, we also detected Wolbachia, nontarget species, and heteroplasmic sequences. Next‐generation sequencing is of great value because of its protocol simplicity, greatly reduced cost per barcode read, faster throughout and added information content.     

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.     

Fast and cost‐effective single nucleotide polymorphism (SNP) detection in the absence of a reference genome using semideep next‐generation Random Amplicon Sequencing (RAMseq)

Biodiversity has suffered a dramatic global decline during the past decades, and monitoring tools are urgently needed providing data for the development and evaluation of conservation efforts both on a species and on a genetic level. However, in wild species, the assessment of genetic diversity is often hampered by the lack of suitable genetic markers. In this article, we present Random Amplicon Sequencing (RAMseq), a novel approach for fast and cost‐effective detection of single nucleotide polymorphisms (SNPs) in nonmodel species by semideep sequencing of random amplicons. By applying RAMseq to the Eurasian otter (Lutra lutra), we identified 238 putative SNPs after quality filtering of all candidate loci and were able to validate 32 of 77 loci tested. In a second step, we evaluated the genotyping performance of these SNP loci in noninvasive samples, one of the most challenging genotyping applications, by comparing it with genotyping results of the same faecal samples at microsatellite markers. We compared (i) polymerase chain reaction (PCR) success rate, (ii) genotyping errors and (iii) Mendelian inheritance (population parameters). SNPs produced a significantly higher PCR success rate (75.5% vs. 65.1%) and lower mean allelic error rate (8.8% vs. 13.3%) than microsatellites, but showed a higher allelic dropout rate (29.7% vs. 19.8%). Genotyping results showed no deviations from Mendelian inheritance in any of the SNP loci. Hence, RAMseq appears to be a valuable tool for the detection of genetic markers in nonmodel species, which is a common challenge in conservation genetic studies.     

Biochar alters the soil microbiome and soil function: results of next‐generation amplicon sequencing across Europe

Wide‐scale application of biochar to soil has been suggested as a mechanism to offset increases in CO₂ emissions through the long‐term sequestration of a carbon rich and inert substance to the soil, but the implications of this for soil diversity and function remain to be determined. Biochar is capable of inducing changes in soil bacterial communities, but the exact impacts of its application are poorly understood. Using three European sites [UK SRC, short rotation coppice, French grassland (FR) and Italian SRF, short rotation forestry (IT)] treated with identical biochar applications, we undertook 16S and ITS amplicon DNA sequencing. In addition, we carried out assessments of community change over time and N and P mobilization in the UK. Significant changes in bacterial and community structure occurred due to treatment, although the nature of the changes varied by site. STAMP differential abundance analysis showed enrichment of Gemmatimonadete and Acidobacteria in UK biochar plots 1 year after application, whilst control plots exhibited enriched Gemmataceae, Isosphaeraceae and Koribacteraceae. Increased mobility of ammonium and phosphates was also detected after 1 year, coupled with a shift from acid to alkaline phosphomonoesterase activity, which may suggest an ecological and functional shift towards a more copiotrophic ecology. Italy also exhibited enrichments, in both the Proteobacteria (driven by an increase in the order Rhizobiales) and the Gemmatimonadetes. No significant change in the abundance of individual taxa was noted in FR, although a small significant change in unweighted UNIFRAC occurred, indicating variation in the identities of taxa present due to treatment. Fungal β diversity was affected by treatment in IT and FR, but was unaffected in UK samples. The effects of time and site were greater than that of biochar application in UK samples. Overall, this report gives a tantalizing view of the soil microbiome at several sites across Europe and suggests that although application of biochar has significant effects on microbial communities, these may be small compared with the highly variable soil microbiome that is found in different soils and changes with time.     

Museum genomics: low-cost and high-accuracy genetic data from historical specimens

Natural history collections are unparalleled repositories of geographical and temporal variation in faunal conditions. Molecular studies offer an opportunity to uncover much of this variation; however, genetic studies of historical museum specimens typically rely on extracting highly degraded and chemically modified DNA samples from skins, skulls or other dried samples. Despite this limitation, obtaining short fragments of DNA sequences using traditional PCR amplification of DNA has been the primary method for genetic study of historical specimens. Few laboratories have succeeded in obtaining genome-scale sequences from historical specimens and then only with considerable effort and cost. Here, we describe a low-cost approach using high-throughput next-generation sequencing to obtain reliable genome-scale sequence data from a traditionally preserved mammal skin and skull using a simple extraction protocol. We show that single-nucleotide polymorphisms (SNPs) from the genome sequences obtained independently from the skin and from the skull are highly repeatable compared to a reference genome.     

Population dynamics of a natural red deer population over 200 years detected via substantial changes of genetic variation

Most large mammals have constantly been exposed to anthropogenic influence over decades or even centuries. Because of their long generation times and lack of sampling material, inferences of past population genetic dynamics, including anthropogenic impacts, have only relied on the analysis of the structure of extant populations. Here, we investigate for the first time the change in the genetic constitution of a natural red deer population over two centuries, using up to 200‐year‐old antlers (30 generations) stored in trophy collections. To the best of our knowledge, this is the oldest DNA source ever used for microsatellite population genetic analyses. We demonstrate that government policy and hunting laws may have strong impacts on populations that can lead to unexpectedly rapid changes in the genetic constitution of a large mammal population. A high ancestral individual polymorphism seen in an outbreeding population (1813–1861) was strongly reduced in descendants (1923–1940) during the mid‐19th and early 20th century by genetic bottlenecks. Today (2011), individual polymorphism and variance among individuals is increasing in a constant‐sized (managed) population. Differentiation was high among periods (F_ST > ***); consequently, assignment tests assigned individuals to their own period with >85% probability. In contrast to the high variance observed at nuclear microsatellite loci, mtDNA (D‐loop) was monomorphic through time, suggesting that male immigration dominates the genetic evolution in this population.     

Robbery in progress: Historical museum collections bring to light a mitochondrial capture within a bird species widespread across southern Australia,the Copperback Quail‐thrush Cinclosoma clarum

We surveyed mitochondrial, autosomal, and Z chromosome diversity within and between the Copperback Quail‐thrush Cinclosoma clarum and Chestnut Quail‐thrush C. castanotum, which together span the arid and semi‐arid zones of southern Australia, and primarily from specimens held in museum collections. We affirm the recent taxonomic separation of the two species and then focus on diversity within the more widespread of the two species, C. clarum. To guide further study of the system and what it offers to understanding the genomics of the differentiation and speciation processes, we develop and present a hypothesis to explain mitonuclear discordance that emerged in ourdata. Following a period of historical allopatry, secondary contact has resulted in an eastern mitochondrial genome replacing the western mitochondrial genome in western populations. This is predicted under a population‐level invasion in the opposite direction, that of the western population invading the range of the eastern one. Mitochondrial captures can be driven by neutral, demographic processes, or adaptive mechanisms, and we favor the hypothesized capture being driven by neutral means. We cannot fully reject the adaptive process but suggest how these alternatives may be further tested. We acknowledge an alternative hypothesis, which finds some support in phenotypic data published elsewhere, namely that outcomes of secondary contact have been more complex than our current genomic data suggest. Discriminating and reconciling these two alternative hypotheses, which may not be mutually exclusive, could be tested with closer sampling at levels of population, individual, and nucleotide than has so far been possible. This would be further aided by knowledge of the genetic basis to phenotypic variation described elsewhere.     

Development of genomic resources for Nothofagus species using next‐generation sequencing data

Using next‐generation sequencing, we developed the first whole‐genome resources for two hybridizing Nothofagus species of the Patagonian forests that crucially lack genomic data, despite their ecological and industrial value. A de novo assembly strategy combining base quality control and optimization of the putative chloroplast gene map yielded ~32 000 contigs from 43% of the reads produced. With 12.5% of assembled reads, we covered ~96% of the chloroplast genome and ~70% of the mitochondrial gene content, providing functional and structural annotations for 112 and 52 genes, respectively. Functional annotation was possible on 15% of the contigs, with ~1750 potentially novel nuclear genes identified for Nothofagus species. We estimated that the new resources (13.41 Mb in total) included ~4000 gene regions representing ~6.5% of the expected genic partition of the genome, the remaining contigs potentially being nongenic DNA. A high‐quality single nucleotide polymorphisms resource was developed by comparing various filtering methods, and preliminary results indicate a strong conservation of cpDNA genomes in contrast to numerous exclusive nuclear polymorphisms in both species. Finally, we characterized 2274 potential simple sequence repeat (SSR) loci, designed primers for 769 of them and validated nine of 29 loci in 42 individuals per species. Nothofagus obliqua had more alleles (4.89) on average than N. nervosa (2.89), 8 SSRs were efficient to discriminate species, and three were successfully transferred in three other Nothofagus species. These resources will greatly help for future inferences of demographic, adaptive and hybridizing events in Nothofagus species, and for conserving and managing natural populations.     

Sequence capture and next‐generation sequencing of ultraconserved elements in a large‐genome salamander

Amidst the rapid advancement in next‐generation sequencing (NGS) technology over the last few years, salamanders have been left behind. Salamanders have enormous genomes—up to 40 times the size of the human genome—and this poses challenges to generating NGS data sets of quality and quantity similar to those of other vertebrates. However, optimization of laboratory protocols is time‐consuming and often cost prohibitive, and continued omission of salamanders from novel phylogeographic research is detrimental to species facing decline. Here, we use a salamander endemic to the southeastern United States, Plethodon serratus, to test the utility of an established protocol for sequence capture of ultraconserved elements (UCEs) in resolving intraspecific phylogeographic relationships and delimiting cryptic species. Without modifying the standard laboratory protocol, we generated a data set consisting of over 600 million reads for 85 P. serratus samples. Species delimitation analyses support recognition of seven species within P. serratus sensu lato, and all phylogenetic relationships among the seven species are fully resolved under a coalescent model. Results also corroborate previous data suggesting nonmonophyly of the Ouachita and Louisiana regions. Our results demonstrate that established UCE protocols can successfully be used in phylogeographic studies of salamander species, providing a powerful tool for future research on evolutionary history of amphibians and other organisms with large genomes.     

Cost‐effective development of highly polymorphic microsatellite in Japanese quail facilitated by next‐generation sequencing

Next‐generation sequencing technologies permit rapid and cost‐effective identification of numerous putative microsatellite loci. Here, from the genome sequences of Japanese quail, we developed microsatellite markers containing dinucleotide repeats and employed these for characterisation of genetic diversity and population structure. A total of 385 individuals from 12 experimental and one wild‐derived Japanese quail lines were genotyped with newly developed autosomal markers. The maximum number of alleles, expected heterozygosity and polymorphic information content (PIC) per locus were 10, 0.80 and 0.77 respectively. Approximately half of the markers were highly informative (PIC ≥ 0.50). The mean number of alleles per locus and observed heterozygosity within a line were in the range of 1.3–4.1 and 0.11–0.53 respectively. Compared with the wild‐derived line, genetic diversity levels were low in the experimental lines. Genetic differentiation (F_ST) between all pairs of the lines ranged from 0.13 to 0.83. Genetic clustering analyses based on multilocus genotypes of individuals showed that most individuals formed clearly defined clusters corresponding to the origins of the lines. These results suggest that Japanese quail experimental lines are highly structured. Microsatellite markers developed in this study may be effective for future genetic studies of Japanese quail.     

Inferring the mode of origin of polyploid species from next‐generation sequence data

Many eukaryote organisms are polyploid. However, despite their importance, evolutionary inference of polyploid origins and modes of inheritance has been limited by a need for analyses of allele segregation at multiple loci using crosses. The increasing availability of sequence data for nonmodel species now allows the application of established approaches for the analysis of genomic data in polyploids. Here, we ask whether approximate Bayesian computation (ABC), applied to realistic traditional and next‐generation sequence data, allows correct inference of the evolutionary and demographic history of polyploids. Using simulations, we evaluate the robustness of evolutionary inference by ABC for tetraploid species as a function of the number of individuals and loci sampled, and the presence or absence of an outgroup. We find that ABC adequately retrieves the recent evolutionary history of polyploid species on the basis of both old and new sequencing technologies. The application of ABC to sequence data from diploid and polyploid species of the plant genus Capsella confirms its utility. Our analysis strongly supports an allopolyploid origin of C. bursa‐pastoris about 80 000 years ago. This conclusion runs contrary to previous findings based on the same data set but using an alternative approach and is in agreement with recent findings based on whole‐genome sequencing. Our results indicate that ABC is a promising and powerful method for revealing the evolution of polyploid species, without the need to attribute alleles to a homeologous chromosome pair. The approach can readily be extended to more complex scenarios involving higher ploidy levels.