GUT MICROBIOME DIVERSITY OF THREE RHINOCEROS SPECIES IN EUROPEAN ZOOS.

The wild rhinoceros populations have declined drastically in the past decades because the rhinoceros are heavily hunted for their horns. Zoological institutions aim to conserve rhinoceros populations in captivity, but one of the challenges of ex situ conservation is to provide food sources that resemble those available in the wild. Considering that the mammalian gut microbiota is a pivotal player in their host's health, the gut microbiota of rhinoceros may also play a role in the bioavailability of nutrients. Therefore, this study aims to characterize the fecal microbiome composition of grazing white rhinoceros (WR; Ceratotherium simum) and greater one-horned rhinoceros (GOHR; Rhinoceros unicornis) as well as the browsing black rhinoceros (BR; Diceros bicornis) kept in European zoos. Over the course of 1 yr, 166 fecal samples in total were collected from 9 BR (n = 39), 10 GOHR (n = 56), and 14 WR (n = 71) from 23 zoological institutions. The bacterial composition in the samples was determined using 16S rRNA gene Illumina sequencing. The fecal microbiomes of rhinoceros clustered by species, with BR clustering more distantly from GOHR and WR. Furthermore, the data report clustering of rhinoceros microbiota according to individual rhinoceros and institutional origin, showing that zoological institutions play a significant role in shaping the gut microbiome of rhinoceros species. In addition, BR exhibit a relatively higher microbial diversity than GOHR and WR. BR seem more susceptible to microbial gut changes and appear to have a more diverse microbiome composition among individuals than GOHR and WR. These data expand on the role of gut microbes and can provide baseline data for continued efforts in rhinoceros conservation and health status.

Comparative Genomics Elucidates the Origin of a Supergene Controlling Floral Heteromorphism.

Supergenes are nonrecombining genomic regions ensuring the coinheritance of multiple, coadapted genes. Despite the importance of supergenes in adaptation, little is known on how they originate. A classic example of supergene is the S locus controlling heterostyly, a floral heteromorphism occurring in 28 angiosperm families. In Primula, heterostyly is characterized by the cooccurrence of two complementary, self-incompatible floral morphs and is controlled by five genes clustered in the hemizygous, ca. 300-kb S locus. Here, we present the first chromosome-scale genome assembly of any heterostylous species, that of Primula veris (cowslip). By leveraging the high contiguity of the P. veris assembly and comparative genomic analyses, we demonstrated that the S-locus evolved via multiple, asynchronous gene duplications and independent gene translocations. Furthermore, we discovered a new whole-genome duplication in Ericales that is specific to the Primula lineage. We also propose a mechanism for the origin of S-locus hemizygosity via nonhomologous recombination involving the newly discovered two pairs of CFB genes flanking the S locus. Finally, we detected only weak signatures of degeneration in the S locus, as predicted for hemizygous supergenes. The present study provides a useful resource for future research addressing key questions on the evolution of supergenes in general and the S locus in particular: How do supergenes arise? What is the role of genome architecture in the evolution of complex adaptations? Is the molecular architecture of heterostyly supergenes across angiosperms similar to that of Primula?

Phylotranscriptomics reveals the reticulate evolutionary history of a widespread diatom species complex.

In contrast to surveys based on a few genes that often provide limited taxonomic resolution, transcriptomes provide a wealth of genomic loci that can resolve relationships among taxonomically challenging lineages. Diatoms are a diverse group of aquatic microalgae that includes important bioindicator species and many such lineages. One example is Nitzschia palea, a widespread species complex with several morphologically defined taxonomic varieties, some of which are critical pollution indicators. Morphological differences among the varieties are subtle and phylogenetic studies based on a few genes fail to resolve their evolutionary relationships. We conducted morphometric and transcriptome analyses of 10 Nitzschia palea strains to resolve the relationships among strains and taxonomic varieties. Nitzschia palea was resolved into three clades, one of which corresponds to a group of strains with narrow linear-lanceolate valves. The other morphological group recovered in the shape outline analysis was not monophyletic and consisted of two clades. Gene-tree concordance analyses and phylogenetic network estimations revealed patterns of incomplete lineage sorting and gene flow between intraspecific lineages. We detected reticulated evolutionary patterns among lineages with different morphologies, resulting in a putative recent hybrid. Our study shows that phylogenomic analyses of unlinked nuclear loci, complemented with morphometrics, can resolve complex evolutionary histories of recently diverged species complexes.

A pseudomolecule-scale genome assembly of the liverwort Marchantia polymorpha.

Marchantia polymorpha has recently become a prime model for cellular, evo-devo, synthetic biological, and evolutionary investigations. We present a pseudomolecule-scale assembly of the M. polymorpha genome, making comparative genome structure analysis and classical genetic mapping approaches feasible. We anchored 88% of the M. polymorpha draft genome to a high-density linkage map resulting in eight pseudomolecules. We found that the overall genome structure of M. polymorpha is in some respects different from that of the model moss Physcomitrella patens. Specifically, genome collinearity between the two bryophyte genomes and vascular plants is limited, suggesting extensive rearrangements since divergence. Furthermore, recombination rates are greatest in the middle of the chromosome arms in M. polymorpha like in most vascular plant genomes, which is in contrast with P. patens where recombination rates are evenly distributed along the chromosomes. Nevertheless, some other properties of the genome are shared with P. patens. As in P. patens, DNA methylation in M. polymorpha is spread evenly along the chromosomes, which is in stark contrast with the angiosperm model Arabidopsis thaliana, where DNA methylation is strongly enriched at the centromeres. Nevertheless, DNA methylation and recombination rate are anticorrelated in all three species. Finally, M. polymorpha and P. patens centromeres are of similar structure and marked by high abundance of retroelements unlike in vascular plants. Taken together, the highly contiguous genome assembly we present opens unexplored avenues for M. polymorpha research by linking the physical and genetic maps, making novel genomic and genetic analyses, including map-based cloning, feasible.

NCBI-compliant genome submissions: tips and tricks to save time and money.

Genome sequences nowadays play a central role in molecular biology and bioinformatics. These sequences are shared with the scientific community through sequence databases. The sequence repositories of the International Nucleotide Sequence Database Collaboration (INSDC, comprising GenBank, ENA and DDBJ) are the largest in the world. Preparing an annotated sequence in such a way that it will be accepted by the database is challenging because many validation criteria apply. In our opinion, it is an undesirable situation that researchers who want to submit their sequence need either a lot of experience or help from partners to get the job done. To save valuable time and money, we list a number of recommendations for people who want to submit an annotated genome to a sequence database, as well as for tool developers, who could help to ease the process.

Homeologs of the Nicotiana benthamiana Antiviral ARGONAUTE1 Show Different Susceptibilities to microRNA168-Mediated Control.

The plant ARGONAUTE1 protein (AGO1) is a central functional component of the posttranscriptional regulation of gene expression and the RNA silencing-based antiviral defense. By genomic and molecular approaches, we here reveal the presence of two homeologs of the AGO1-like gene in Nicotiana benthamiana, NbAGO1-1H and NbAGO1-1L. Both homeologs retain the capacity to transcribe messenger RNAs (mRNAs), which mainly differ in one 18-nucleotide insertion/deletion (indel). The indel does not modify the frame of the open reading frame, and it is located eight nucleotides upstream of the target site of a microRNA, miR168, which is an important modulator of AGO1 expression. We demonstrate that there is a differential accumulation of the two NbAGO1-1 homeolog mRNAs at conditions where miR168 is up-regulated, such as during a tombusvirus infection. The data reported suggest that the indel affects the miR168-guided regulation of NbAGO1 mRNA. The two AGO1 homeologs show full functionality in reconstituted, catalytically active RNA-induced silencing complexes following the incorporation of small interfering RNAs. Virus-induced gene silencing experiments suggest a specific involvement of the NbAGO1 homeologs in symptom development. The results provide an example of the diversity of microRNA target regions in NbAGO1 homeolog genes, which has important implications for improving resilience measures of the plant during viral infections.

The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system.

Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection.

SSPACE-LongRead: scaffolding bacterial draft genomes using long read sequence information.

BACKGROUND: The recent introduction of the Pacific Biosciences RS single molecule sequencing technology has opened new doors to scaffolding genome assemblies in a cost-effective manner. The long read sequence information is promised to enhance the quality of incomplete and inaccurate draft assemblies constructed from Next Generation Sequencing (NGS) data. RESULTS: Here we propose a novel hybrid assembly methodology that aims to scaffold pre-assembled contigs in an iterative manner using PacBio RS long read information as a backbone. On a test set comprising six bacterial draft genomes, assembled using either a single Illumina MiSeq or Roche 454 library, we show that even a 50× coverage of uncorrected PacBio RS long reads is sufficient to drastically reduce the number of contigs. Comparisons to the AHA scaffolder indicate our strategy is better capable of producing (nearly) complete bacterial genomes. CONCLUSIONS: The current work describes our SSPACE-LongRead software which is designed to upgrade incomplete draft genomes using single molecule sequences. We conclude that the recent advances of the PacBio sequencing technology and chemistry, in combination with the limited computational resources required to run our program, allow to scaffold genomes in a fast and reliable manner.

Genotyping by sequencing for estimating relative abundances of diatom taxa in mock communities.

BACKGROUND: Diatoms are present in all waters and are highly sensitive to pollution gradients. Therefore, they are ideal bioindicators for water quality assessment. Current indices used in these applications are based on identifying diatom species and counting their abundances using traditional light microscopy. Several molecular techniques have been developed to help automate different steps of this process, but obtaining reliable estimates of diatom community composition and species abundance remains challenging. RESULTS: Here, we evaluated a recently developed quantification method based on Genotyping by Sequencing (GBS) for the first time in diatoms to estimate the relative abundances within a species complex. For this purpose, a reference database comprised of thousands of genomic DNA clusters was generated from cultures of Nitzschia palea. The sequencing reads from calibration and mock samples were mapped against this database for parallel quantification. We sequenced 25 mock diatom communities containing up to five taxa per sample in different abundances. Taxon abundances in these communities were also quantified by a diatom expert using manual counting of cells on light microscopic slides. The relative abundances of strains across mock samples were over- or under-estimated by the manual counting method, and a majority of mock samples had stronger correlations using GBS. Moreover, one previously recognized putative hybrid had the largest number of false positive detections demonstrating the limitation of the manual counting method when morphologically similar and/or phylogenetically close taxa are analyzed. CONCLUSIONS: Our results suggest that GBS is a reliable method to estimate the relative abundances of the N. palea taxa analyzed in this study and outperformed traditional light microscopy in terms of accuracy. GBS provides increased taxonomic resolution compared to currently available quantitative molecular approaches, and it is more scalable in the number of species that can be analyzed in a single run. Hence, this is a significant step forward in developing automated, high-throughput molecular methods specifically designed for the quantification of [diatom] communities for freshwater quality assessments.

The complete mitogenome of Cylindrus obtusus (Helicidae, Ariantinae) using Illumina next generation sequencing.

BACKGROUND: This study describes how the complete mitogenome of a terrestrial snail, Cylindrus obtusus (Draparnaud, 1805) was sequenced without PCRs from a collection specimen that had been in 70% ethanol for 8 years. The mitogenome was obtained with Illumina GAIIx shot gun sequencing. Although the used specimen was collected relatively recently and kept in a DNA-friendly preservative (not formalin as frequently used with old museum specimens), we believe that the exclusion of PCRs as facilitated by NGS (Next Generation Sequencing) removes a great obstacle in DNA sequencing of collection specimens. A brief comparison is made between our Illumina GAIIx approach and a similar study that made use of the Roche 454-FLX platform. RESULTS: The mtDNA sequence of C. obtusus is 14,610 bases in length (about 0.5 kb larger than other stylommatophoran mitogenomes reported hitherto) and contains the 37 genes (13 protein coding genes, two rRNAs and 22 tRNAs) typical for metazoans. Except for a swap between the position of tRNA-Pro and tRNA-Ala, the gene arrangement of C. obtusus is identical to that reported for Cepaea nemoralis. The 'aberrant' rearrangement of tRNA-Thr and COIII compared to that of other Sigmurethra (and the majority of gastropods), is not unique for C. nemoralis (subfamily Helicinae), but is also shown to occur in C. obtusus (subfamily Ariantinae) and might be a synapomorphy for the family Helicidae. CONCLUSIONS: Natural history collections potentially harbor a wealth of information for the field of evolutionary genetics, but it can be difficult to amplify DNA from such specimens (due to DNA degradation for instance). Because NGS techniques do not rely on primer-directed amplification (PCR) and allow DNA to be fragmented (DNA gets sheared during library preparation), NGS could be a valuable tool for retrieving DNA sequence data from such specimens. A comparison between Illumina GAIIx and the Roche 454 platform suggests that the former might be more suited for de novo sequencing of mitogenomes.

Scaffolding pre-assembled contigs using SSPACE.

SUMMARY: De novo assembly tools play a main role in reconstructing genomes from next-generation sequencing (NGS) data and usually yield a number of contigs. Using paired-read sequencing data it is possible to assess the order, distance and orientation of contigs and combine them into so-called scaffolds. Although the latter process is a crucial step in finishing genomes, scaffolding algorithms are often built-in functions in de novo assembly tools and cannot be independently controlled. We here present a new tool, called SSPACE, which is a stand-alone scaffolder of pre-assembled contigs using paired-read data. Main features are: a short runtime, multiple library input of paired-end and/or mate pair datasets and possible contig extension with unmapped sequence reads. SSPACE shows promising results on both prokaryote and eukaryote genomic testsets where the amount of initial contigs was reduced by at least 75%.

Comparative transcriptomics reveals commonalities and differences in the genetic underpinnings of a floral dimorphism.

Distyly, a floral dimorphism associated with heteromorphic self-incompatibility and controlled by the S-locus supergene, evolved independently multiple times. Comparative analyses of the first transcriptome atlas for the main distyly model, Primula veris, with other distylous species produced the following findings. A set of 53 constitutively expressed genes in P. veris did not include any of the housekeeping genes commonly used to normalize gene expression in qPCR experiments. The S-locus gene CYPT acquired its role in controlling style elongation via a change in expression profile. Comparison of genes differentially expressed between floral morphs revealed that brassinosteroids and auxin are the main hormones controlling style elongation in P. veris and Fagopyrum esculentum, respectively. Furthermore, shared biochemical pathways might underlie the expression of distyly in the distantly related P. veris, F. esculentum and Turnera subulata, suggesting a degree of correspondence between evolutionary convergence at phenotypic and molecular levels. Finally, we provide the first evidence supporting the previously proposed hypothesis that distyly supergenes of distantly related species evolved via the recruitment of genes related to the phytochrome-interacting factor (PIF) signaling network. To conclude, this is the first study that discovered homologous genes involved in the control of distyly in distantly related taxa.

Development of a reproducible small intestinal microbiota model and its integration into the SHIME®-system, a dynamic in vitro gut model.

The human gastrointestinal tract consists of different regions, each characterized by a distinct physiology, anatomy, and microbial community. While the colonic microbiota has received a lot of attention in recent research projects, little is known about the small intestinal microbiota and its interactions with ingested compounds, primarily due to the inaccessibility of this region in vivo. This study therefore aimed to develop and validate a dynamic, long-term simulation of the ileal microbiota using the SHIME®-technology. Essential parameters were identified and optimized from a screening experiment testing different inoculation strategies, nutritional media, and environmental parameters over an 18-day period. Subjecting a synthetic bacterial consortium to the selected conditions resulted in a stable microbiota that was representative in terms of abundance [8.81 ± 0.12 log (cells/ml)], composition and function. Indeed, the observed community mainly consisted of the genera Streptococcus, Veillonella, Enterococcus, Lactobacillus, and Clostridium (qPCR and 16S rRNA gene targeted Illumina sequencing), while nutrient administration boosted lactate production followed by cross-feeding interactions towards acetate and propionate. Furthermore, similarly as in vivo, bile salts were only partially deconjugated and only marginally converted into secondary bile salts. After confirming reproducibility of the small intestinal microbiota model, it was integrated into the established M-SHIME® where it further increased the compositional relevance of the colonic community. This long-term in vitro model provides a representative simulation of the ileal bacterial community, facilitating research of the ileum microbiota dynamics and activity when, for example, supplemented with microbial or diet components. Furthermore, integration of this present in vitro simulation increases the biological relevance of the current M-SHIME® technology.

Correlated substitution analysis and the prediction of amino acid structural contacts.

It has long been suspected that analysis of correlated amino acid substitutions should uncover pairs or clusters of sites that are spatially proximal in mature protein structures. Accordingly, methods based on different mathematical principles such as information theory, correlation coefficients and maximum likelihood have been developed to identify co-evolving amino acids from multiple sequence alignments. Sets of pairs of sites whose behaviour is identified by these methods as correlated are often significantly enriched in pairs of spatially proximal residues. However, relatively high levels of false-positive predictions typically render such methods, in isolation, of little use in the ab initio prediction of protein structure. Misleading signal (or problems with the estimation of significance levels) can be caused by phylogenetic correlations between homologous sequences and from correlation due to factors other than spatial proximity (for example, correlation of sites which are not spatially close but which are involved in common functional properties of the protein). In recent years, several workers have suggested that information from correlated substitutions should be combined with other sources of information (secondary structure, solvent accessibility, evolutionary rates) in an attempt to reduce the proportion of false-positive predictions. We review methods for the detection of correlated amino acid substitutions, compare their relative performance in contact prediction and predict future directions in the field.

CGHnormaliter: an iterative strategy to enhance normalization of array CGH data with imbalanced aberrations.

BACKGROUND: Array comparative genomic hybridization (aCGH) is a popular technique for detection of genomic copy number imbalances. These play a critical role in the onset of various types of cancer. In the analysis of aCGH data, normalization is deemed a critical pre-processing step. In general, aCGH normalization approaches are similar to those used for gene expression data, albeit both data-types differ inherently. A particular problem with aCGH data is that imbalanced copy numbers lead to improper normalization using conventional methods. RESULTS: In this study we present a novel method, called CGHnormaliter, which addresses this issue by means of an iterative normalization procedure. First, provisory balanced copy numbers are identified and subsequently used for normalization. These two steps are then iterated to refine the normalization. We tested our method on three well-studied tumor-related aCGH datasets with experimentally confirmed copy numbers. Results were compared to a conventional normalization approach and two more recent state-of-the-art aCGH normalization strategies. Our findings show that, compared to these three methods, CGHnormaliter yields a higher specificity and precision in terms of identifying the 'true' copy numbers. CONCLUSION: We demonstrate that the normalization of aCGH data can be significantly enhanced using an iterative procedure that effectively eliminates the effect of imbalanced copy numbers. This also leads to a more reliable assessment of aberrations. An R-package containing the implementation of CGHnormaliter is available at http://www.ibi.vu.nl/programs/cghnormaliterwww.

PRALINETM: a strategy for improved multiple alignment of transmembrane proteins.

MOTIVATION: Membrane-bound proteins are a special class of proteins. The regions that insert into the cell-membrane have a profoundly different hydrophobicity pattern compared with soluble proteins. Multiple alignment techniques use scoring schemes tailored for sequences of soluble proteins and are therefore in principle not optimal to align membrane-bound proteins. RESULTS: Transmembrane (TM) regions in protein sequences can be reliably recognized using state-of-the-art sequence prediction techniques. Furthermore, membrane-specific scoring matrices are available. We have developed a new alignment method, called PRALINETM, which integrates these two features to enhance multiple sequence alignment. We tested our algorithm on the TM alignment benchmark set by Bahr et al. (2001), and showed that the quality of TM alignments can be significantly improved compared with the quality produced by a standard multiple alignment technique. The results clearly indicate that the incorporation of these new elements into current state-of-the-art alignment methods is crucial for optimizing the alignment of TM proteins. AVAILABILITY: A webserver is available at http://www.ibi.vu.nl/programs/pralinewww.

Sequence harmony: detecting functional specificity from alignments.

Multiple sequence alignments are often used for the identification of key specificity-determining residues within protein families. We present a web server implementation of the Sequence Harmony (SH) method previously introduced. SH accurately detects subfamily specific positions from a multiple alignment by scoring compositional differences between subfamilies, without imposing conservation. The SH web server allows a quick selection of subtype specific sites from a multiple alignment given a subfamily grouping. In addition, it allows the predicted sites to be directly mapped onto a protein structure and displayed. We demonstrate the use of the SH server using the family of plant mitochondrial alternative oxidases (AOX). In addition, we illustrate the usefulness of combining sequence and structural information by showing that the predicted sites are clustered into a few distinct regions in an AOX homology model. The SH web server can be accessed at www.ibi.vu.nl/programs/seqharmwww.

On the Role of Bioinformatics and Data Science in Industrial Microbiome Applications.

Advances in sequencing and computational biology have drastically increased our capability to explore the taxonomic and functional compositions of microbial communities that play crucial roles in industrial processes. Correspondingly, commercial interest has risen for applications where microbial communities make important contributions. These include food production, probiotics, cosmetics, and enzyme discovery. Other commercial applications include software that takes the user's gut microbiome data as one of its inputs and outputs evidence-based, automated, and personalized diet recommendations for balanced blood sugar levels. These applications pose several bioinformatic and data science challenges that range from requiring strain-level resolution in community profiles to the integration of large datasets for predictive machine learning purposes. In this perspective, we provide our insights on such challenges by touching upon several industrial areas, and briefly discuss advances and future directions of bioinformatics and data science in microbiome research.

Complete Genome Sequence of Malassezia restricta CBS 7877, an Opportunist Pathogen Involved in Dandruff and Seborrheic Dermatitis.

Malassezia restricta, one of the predominant basidiomycetous yeasts present on human skin, is involved in scalp disorders. Here, we report the complete genome sequence of the lipophilic Malassezia restricta CBS 7877 strain, which will facilitate the study of the mechanisms underlying its commensal and pathogenic roles within the skin microbiome.

The meaning of alignment: lessons from structural diversity.

BACKGROUND: Protein structural alignment provides a fundamental basis for deriving principles of functional and evolutionary relationships. It is routinely used for structural classification and functional characterization of proteins and for the construction of sequence alignment benchmarks. However, the available techniques do not fully consider the implications of protein structural diversity and typically generate a single alignment between sequences. RESULTS: We have taken alternative protein crystal structures and generated simulation snapshots to explicitly investigate the impact of structural changes on the alignments. We show that structural diversity has a significant effect on structural alignment. Moreover, we observe alignment inconsistencies even for modest spatial divergence, implying that the biological interpretation of alignments is less straightforward than commonly assumed. A salient example is the GroES 'mobile loop' where sub-Angstrom variations give rise to contradictory sequence alignments. CONCLUSION: A comprehensive treatment of ambiguous alignment regions is crucial for further development of structural alignment applications and for the representation of alignments in general. For this purpose we have developed an on-line database containing our data and new ways of visualizing alignment inconsistencies, which can be found at http://www.ibi.vu.nl/databases/stralivari.