De novo sequencing, assembly and functional annotation of Armillaria borealis genome.

BACKGROUND: Massive forest decline has been observed almost everywhere as a result of negative anthropogenic and climatic effects, which can interact with pests, fungi and other phytopathogens and aggravate their effects. Climatic changes can weaken trees and make fungi, such as Armillaria more destructive. Armillaria borealis (Marxm. & Korhonen) is a fungus from the Physalacriaceae family (Basidiomycota) widely distributed in Eurasia, including Siberia and the Far East. Species from this genus cause the root white rot disease that weakens and often kills woody plants. However, little is known about ecological behavior and genetics of A. borealis. According to field research data, A. borealis is less pathogenic than A. ostoyae, and its aggressive behavior is quite rare. Mainly A. borealis behaves as a secondary pathogen killing trees already weakened by other factors. However, changing environment might cause unpredictable effects in fungus behavior. RESULTS: The de novo genome assembly and annotation were performed for the A. borealis species for the first time and presented in this study. The A. borealis genome assembly contained ~ 68 Mbp and was comparable with ~ 60 and ~ 79.5 Mbp for the A. ostoyae and A. mellea genomes, respectively. The N50 for contigs equaled 50,544 bp. Functional annotation analysis revealed 21,969 protein coding genes and provided data for further comparative analysis. Repetitive sequences were also identified. The main focus for further study and comparative analysis will be on the enzymes and regulatory factors associated with pathogenicity. CONCLUSIONS: Pathogenic fungi such as Armillaria are currently one of the main problems in forest conservation. A comprehensive study of these species and their pathogenicity is of great importance and needs good genomic resources. The assembled genome of A. borealis presented in this study is of sufficiently good quality for further detailed comparative study on the composition of enzymes in other Armillaria species. There is also a fundamental problem with the identification and classification of species of the Armillaria genus, where the study of repetitive sequences in the genomes of basidiomycetes and their comparative analysis will help us identify more accurately taxonomy of these species and reveal their evolutionary relationships.

Stepwise large genome assembly approach: a case of Siberian larch (Larix sibirica Ledeb).

BACKGROUND: De novo assembling of large genomes, such as in conifers (~ 12-30 Gbp), which also consist of ~ 80% of repetitive DNA, is a very complex and computationally intense endeavor. One of the main problems in assembling such genomes lays in computing limitations of nucleotide sequence assembly programs (DNA assemblers). As a rule, modern assemblers are usually designed to assemble genomes with a length not exceeding the length of the human genome (3.24 Gbp). Most assemblers cannot handle the amount of input sequence data required to provide sufficient coverage needed for a high-quality assembly. RESULTS: An original stepwise method of de novo assembly by parts (sets), which allows to bypass the limitations of modern assemblers associated with a huge amount of data being processed, is presented in this paper. The results of numerical assembling experiments conducted using the model plant Arabidopsis thaliana, Prunus persica (peach) and four most popular assemblers, ABySS, SOAPdenovo, SPAdes, and CLC Assembly Cell, showed the validity and effectiveness of the proposed stepwise assembling method. CONCLUSION: Using the new stepwise de novo assembling method presented in the paper, the genome of Siberian larch, Larix sibirica Ledeb. (12.34 Gbp) was completely assembled de novo by the CLC Assembly Cell assembler. It is the first genome assembly for larch species in addition to only five other conifer genomes sequenced and assembled for Picea abies, Picea glauca, Pinus taeda, Pinus lambertiana, and Pseudotsuga menziesii var. menziesii.

De novo assembling and primary analysis of genome and transcriptome of gray whale Eschrichtius robustus.

BACKGROUND: Gray whale, Eschrichtius robustus (E. robustus), is a single member of the family Eschrichtiidae, which is considered to be the most primitive in the class Cetacea. Gray whale is often described as a "living fossil". It is adapted to extreme marine conditions and has a high life expectancy (77 years). The assembly of a gray whale genome and transcriptome will allow to carry out further studies of whale evolution, longevity, and resistance to extreme environment. RESULTS: In this work, we report the first de novo assembly and primary analysis of the E. robustus genome and transcriptome based on kidney and liver samples. The presented draft genome assembly is complete by 55% in terms of a total genome length, but only by 24% in terms of the BUSCO complete gene groups, although 10,895 genes were identified. Transcriptome annotation and comparison with other whale species revealed robust expression of DNA repair and hypoxia-response genes, which is expected for whales. CONCLUSIONS: This preliminary study of the gray whale genome and transcriptome provides new data to better understand the whale evolution and the mechanisms of their adaptation to the hypoxic conditions.

Annotation of Siberian Larch (Larix sibirica Ledeb.) Nuclear Genome-One of the Most Cold-Resistant Tree Species in the Only Deciduous GENUS in Pinaceae.

The recent release of the nuclear, chloroplast and mitochondrial genome assemblies of Siberian larch (Larix sibirica Ledeb.), one of the most cold-resistant tree species in the only deciduous genus of Pinaceae, with seasonal senescence and a rot-resistant valuable timber widely used in construction, greatly contributed to the development of genomic resources for the larch genus. Here, we present an extensive repeatome analysis and the first annotation of the draft nuclear Siberian larch genome assembly. About 66% of the larch genome consists of highly repetitive elements (REs), with the likely wave of retrotransposons insertions into the larch genome estimated to occur 4-5 MYA. In total, 39,370 gene models were predicted, with 87% of them having homology to the Arabidopsis-annotated proteins and 78% having at least one GO term assignment. The current state of the genome annotations allows for the exploration of the gymnosperm and angiosperm species for relative gene abundance in different functional categories. Comparative analysis of functional gene categories across different angiosperm and gymnosperm species finds that the Siberian larch genome has an overabundance of genes associated with programmed cell death (PCD), autophagy, stress hormone biosynthesis and regulatory pathways; genes that may play important roles in seasonal senescence and stress response to extreme cold in larch. Despite being incomplete, the draft assemblies and annotations of the conifer genomes are at a point of development where they now represent a valuable source for further genomic, genetic and population studies.

Diversity and shifts of the bacterial community associated with Baikal sponge mass mortalities.

The disease of freshwater sponges was first discovered in 2011, when pink samples were found in the Central Basin of Lake Baikal. Subsequently, the visible signs of the disease have changed, and now sponges appear with various symptoms of damage to the body, such as discoloration, tissue necrosis, the formation of brown patches and dirty-purple biofilms on some branches. These signs of the disease are accompanied by the mass death of sponges. We identified differences in microbiomes by sequencing 16S rRNA genes and found changes in the consortium of microorganisms of freshwater Baikal sponges. We found that the observed imbalance in the studied microbial communities of diseased sponges is caused by several different conditionally pathogenic microorganisms that increase their negative effect by acting together and in concert, which leads to the death of photosynthetic microalgae and sponges. Sponges are an important component of coastal communities, and the massive loss of sponges can obviously affect the structure of benthic communities and the purity of water.

The rearrangement of motif F in the flavivirus RNA-directed RNA polymerase.

In the flavivirus genus, the non-structural protein NS5 plays a central role in RNA viral replication and constitutes a major target for drug discovery. One of the prime challenges in the study of NS5 protein is to investigate the interplay between the two protein domains, namely, the RNA-dependent RNA polymerase (RdRp) domain and the methyltransferase (MTase) domain. These investigations could clarify the multiple roles of NS5 protein in the virus life cycle. Here we present the results of sequence analyses and structural bioinformatics studies of NS5 protein, which suggest that the conserved motif F in the NS5 protein could act as a lock which controls the rearrangement of the domains and as a switch in the protein enzymatic activity.

The signs of adaptive mutations identified in the chloroplast genome of the algae endosymbiont of Baikal sponge.

Background: The study of ecosystems of the great lakes is important as observations can be extended to ecosystems of larger scale. The ecological crisis of Lake Baikal needs investigations to discover the molecular mechanisms involved in the crisis. The disease of Baikal sponges is one of the processes resulting in the degradation of the littoral zone of the lake. Methods: The chloroplast genome fragment for the algae endosymbiont of Baikal sponge was assembled from metagenomic sequencing data. The distributions of polymorphic sites were obtained for the genome fragment, separately for samples from healthy sponge, diseased sponge and dead sponge tissues. Results: The comparative analysis of chloroplast genome sequences suggests that the symbiotic algae from Baikal sponge is close to Choricystis genus of unicellular algae. Also, the distributions of polymorphic sites allowed detection of the signs of extensive mutations in the chloroplasts isolated from the diseased sponge tissues. Conclusions: The study demonstrate the particular case of evolution at the molecular level due to the conditions of a severe crisis of a whole ecosystem in Lake Baikal. The detection of adaptive mutations in the chloroplast genome is an important feature which could represent the behavior of an ecosystem in the event of a severe crisis.

Evaluating the use of diversity indices to distinguish between microbial communities with different traits.

Several measures of biodiversity are commonly used to describe microbial communities, analyzed using 16S gene sequencing. A wide range of available experiments on 16S gene sequencing allows us to present a framework for a comparison of various diversity indices. The criterion for the comparison is the statistical significance of the difference in index values for microbial communities with different traits, within the same experiment. The results of the evaluation indicate that Shannon diversity is the most effective measure among the commonly used diversity indices. The results also indicate that, within the present framework, the Gini coefficient as a diversity index is comparable to Shannon diversity, despite the fact that the Gini coefficient, as a diversity estimator, is far less popular in microbiology than several other measures.

Comparative genomics of Vibrio cholerae El Tor strains isolated at epidemic complications in Siberia and at the Far East.

The territory of Siberia and the Far East of Russia is classified as epidemically safe for cholera; however, in the 1970s and 1990s a number of infection importation cases and acute outbreaks associated with the cholera importation were reported. Here, we analyze genomes of four Vibrio cholerae El Tor strains isolated from humans during epidemic complications (imported cases, an outbreak) in the 1990s. The analyzed strains harbor the classical allele of the cholera toxin subunit B gene (ctxB1); thus, belong to genetically altered variants of the El Tor biotype. Analysis of the genomes revealed their high homology with the V. cholerae N16961 reference strain: 85-93 SNPs were identified in the core genome as compared to the reference. The determined features of SNPs in the CTX prophage made it possible to propose the presence of a new subtype - CTX-2a in two strains; the other two strains carried the prophage of CTX-3 type. Results of phylogenetic analysis based on SNP-typing demonstrated that two strains belonged to the second wave, and two - to the early third wave of cholera dissemination in the world. Phylogenetic reconstruction in combination with epidemiological data permitted to trace the origin of the strains and the way of their importation to the Russian Federation directly or through temporary cholera foci.

Genome-wide adaptive complexes to underground stresses in blind mole rats Spalax.

The blind mole rat (BMR), Spalax galili, is an excellent model for studying mammalian adaptation to life underground and medical applications. The BMR spends its entire life underground, protecting itself from predators and climatic fluctuations while challenging it with multiple stressors such as darkness, hypoxia, hypercapnia, energetics and high pathonecity. Here we sequence and analyse the BMR genome and transcriptome, highlighting the possible genomic adaptive responses to the underground stressors. Our results show high rates of RNA/DNA editing, reduced chromosome rearrangements, an over-representation of short interspersed elements (SINEs) probably linked to hypoxia tolerance, degeneration of vision and progression of photoperiodic perception, tolerance to hypercapnia and hypoxia and resistance to cancer. The remarkable traits of the BMR, together with its genomic and transcriptomic information, enhance our understanding of adaptation to extreme environments and will enable the utilization of BMR models for biomedical research in the fight against cancer, stroke and cardiovascular diseases.

NS2B/NS3 protease: allosteric effect of mutations associated with the pathogenicity of tick-borne encephalitis virus.

The sequences of the protease domain of the tick-borne encephalitis (TBE) virus NS3 protein have two amino acid substitutions, 16 R→K and 45 S→F, in the highly pathogenic and poorly pathogenic strains of the virus, respectively. Two models of the NS2B-NS3 protease complex for the highly pathogenic and poorly pathogenic strains of the virus were constructed by homology modeling using the crystal structure of West Nile virus NS2B-NS3 protease as a template; 20 ns molecular dynamic simulations were performed for both models, the trajectories of the dynamic simulations were compared, and the averaged distance between the two models was calculated for each residue. Conformational differences between two models were revealed in the identified pocket. The different conformations of the pocket resulted in different orientations of the NS2B segment located near the catalytic triad. In the model of the highly pathogenic TBE virus the identified pocket had a more open conformation compared to the poorly pathogenic model. We propose that conformational changes in the active protease center, caused by two amino acid substitutions, can influence enzyme functioning and the virulence of the virus.