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The human genome reference assembly is crucial for aligning and analyzing sequence data, and for genome annotation, among other roles. However, the models and analysis assumptions that underlie the current assembly need revising to fully represent human sequence diversity. Improved analysis tools and updated data reporting formats are also required.

Adaptation to specialized diets often requires modifications at both genomic and microbiome levels. We applied a hologenomic approach to the common vampire bat (Desmodus rotundus), one of the only three obligate blood-feeding (sanguivorous) mammals, to study the evolution of its complex dietary adaptation. Specifically, we assembled its high-quality reference genome (scaffold N50=26.9 Mb, contig N50=36.6 Kb) and gut metagenome, and compared them against those of insectivorous, frugivorous, and carnivorous bats. Our analyses showed i) a particular common vampire bat genomic landscape regarding integrated viral elements, ii) a dietary and phylogenetic influence on gut microbiome taxonomic and functional profiles, and iii) that both genetic elements harbor key traits related to the nutritional (e.g. vitamins and lipids shortage) and non-nutritional challenges (e.g. nitrogen waste and osmotic homeostasis) of sanguivory. These findings highlight the value of a holistic study of both host and microbiota when attempting to decipher adaptations underlying radical dietary lifestyles.

As sequencing technologies become more affordable, it is now realistic to propose studying the evolutionary history of virtually any organism on a genomic scale. However, when dealing with non-model organisms it is not always easy to choose the best approach given a specific biological question, a limited budget, and challenging sample material. Furthermore, although recent advances in technology offer unprecedented opportunities for research in non-model organisms, they also demand unprecedented awareness from the researcher regarding the assumptions and limitations of each method. In this review we present an overview of the current sequencing technologies and the methods used in typical high-throughput data analysis pipelines. Subsequently, we contextualize high-throughput DNA sequencing technologies within their applications in non-model organism biology. We include tips regarding managing unconventional sample material, comparative and population genetic approaches that do not require fully assembled genomes, and advice on how to deal with low depth sequencing data.

BackgroundTemperate winters produce extreme energetic challenges for small insectivorous mammals. Some bat species inhabiting locations with mild temperate winters forage during brief inter-torpor normothermic periods of activity. However, the winter diet of bats in mild temperate locations is studied infrequently. Although microscopic analyses of faeces have traditionally been used to characterise bat diet, recently the coupling of PCR with second generation sequencing has offered the potential to further advance our understanding of animal dietary composition and foraging behaviour by allowing identification of a much greater proportion of prey items often with increased taxonomic resolution. We used morphological analysis and Illumina-based second generation sequencing to study the winter diet of Natterer’s bat (Myotis nattereri) and compared the results obtained from these two approaches. For the first time, we demonstrate the applicability of the Illumina MiSeq platform as a data generation source for bat dietary analyses.ResultsFaecal pellets collected from a hibernation site in southern England during two winters (December-March 2009–10 and 2010–11), indicated that M. nattereri forages throughout winter at least in a location with a mild winter climate. Through morphological analysis, arthropod fragments from seven taxonomic orders were identified. A high proportion of these was non-volant (67.9% of faecal pellets) and unexpectedly included many lepidopteran larvae. Molecular analysis identified 43 prey species from six taxonomic orders and confirmed the frequent presence of lepidopteran species that overwinter as larvae.ConclusionsThe winter diet of M. nattereri is substantially different from other times of the year confirming that this species has a wide and adaptable dietary niche. Comparison of DNA derived from the prey to an extensive reference dataset of potential prey barcode sequences permitted fine scale taxonomic resolution of prey species. The high occurrence of non-volant prey suggests that gleaning allows prey capture at low ambient temperatures when the abundance of flying insects may be substantially reduced. Interesting questions arise as to how M. nattereri might successfully locate and capture some of the non-volant prey species encountered in its faeces. The consumption of lepidopteran larvae such as cutworms suggests that M. nattereri eats agricultural pest species.

BackgroundDNA metabarcoding is an approach for identifying multiple taxa in an environmental sample using specific genetic loci and taxa-specific primers. When combined with high-throughput sequencing it enables the taxonomic characterization of large numbers of samples in a relatively time- and cost-efficient manner. One recent laboratory development is the addition of 5′-nucleotide tags to both primers producing double-tagged amplicons and the use of multiple PCR replicates to filter erroneous sequences. However, there is currently no available toolkit for the straightforward analysis of datasets produced in this way.ResultsWe present DAMe, a toolkit for the processing of datasets generated by double-tagged amplicons from multiple PCR replicates derived from an unlimited number of samples. Specifically, DAMe can be used to (i) sort amplicons by tag combination, (ii) evaluate PCR replicates dissimilarity, and (iii) filter sequences derived from sequencing/PCR errors, chimeras, and contamination. This is attained by calculating the following parameters: (i) sequence content similarity between the PCR replicates from each sample, (ii) reproducibility of each unique sequence across the PCR replicates, and (iii) copy number of the unique sequences in each PCR replicate. We showcase the insights that can be obtained using DAMe prior to taxonomic assignment, by applying it to two real datasets that vary in their complexity regarding number of samples, sequencing libraries, PCR replicates, and used tag combinations. Finally, we use a third mock dataset to demonstrate the impact and importance of filtering the sequences with DAMe.ConclusionsDAMe allows the user-friendly manipulation of amplicons derived from multiple samples with PCR replicates built in a single or multiple sequencing libraries. It allows the user to: (i) collapse amplicons into unique sequences and sort them by tag combination while retaining the sample identifier and copy number information, (ii) identify sequences carrying unused tag combinations, (iii) evaluate the comparability of PCR replicates of the same sample, and (iv) filter tagged amplicons from a number of PCR replicates using parameters of minimum length, copy number, and reproducibility across the PCR replicates. This enables an efficient analysis of complex datasets, and ultimately increases the ease of handling datasets from large-scale studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-2064-9) contains supplementary material, which is available to authorized users.

Lions are one of the world’s most iconic megafauna, yet little is known about their temporal and spatial demographic history and population differentiation. We analyzed a genomic dataset of 20 specimens: two ca. 30,000-y-old cave lions (Panthera leo spelaea), 12 historic lions (Panthera leo leo/Panthera leo melanochaita) that lived between the 15th and 20th centuries outside the current geographic distribution of lions, and 6 present-day lions from Africa and India. We found that cave and modern lions shared an ancestor ca. 500,000 y ago and that the 2 lineages likely did not hybridize following their divergence. Within modern lions, we found 2 main lineages that diverged ca. 70,000 y ago, with clear evidence of subsequent gene flow. Our data also reveal a nearly complete absence of genetic diversity within Indian lions, probably due to well-documented extremely low effective population sizes in the recent past. Our results contribute toward the understanding of the evolutionary history of lions and complement conservation efforts to protect the diversity of this vulnerable species.

Saber-toothed cats (Machairodontinae) are among the most widely recognized representatives of the now largely extinct Pleistocene megafauna. However, many aspects of their ecology, evolution, and extinction remain uncertain. Although ancient-DNA studies have led to huge advances in our knowledge of these aspects of many other megafauna species (e.g., mammoths and cave bears), relatively few ancient-DNA studies have focused on saber-toothed cats [1-3], and they have been restricted to short fragments of mitochondrial DNA. Here we investigate the evolutionary history of two lineages of saber-toothed cats (Smilodon and Homotherium) in relation to living carnivores and find that the Machairodontinae form a well-supported clade that is distinct from all living felids. We present partial mitochondrial genomes from one S. populator sample and three Homotherium sp. samples, including the only Late Pleistocene Homotherium sample from Eurasia [4]. We confirm the identification of the unique Late Pleistocene European fossil through ancient-DNA analyses, thus strengthening the evidence that Homotherium occurred in Europe over 200,000 years later than previously believed. This in turn forces a re-evaluation of its demography and extinction dynamics. Within the Machairodontinae, we find a deep divergence between Smilodon and Homotherium (∼18 million years) but limited diversity between the American and European Homotherium specimens. The genetic data support the hypothesis that all Late Pleistocene (or post-Villafrancian) Homotherium should be considered a single species, H. latidens, which was previously proposed based on morphological data [5, 6].