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April 21, 2020  |  

Whole Genome Sequencing of the Mutamouse Model Reveals Strain- and Colony-Level Variation, and Genomic Features of the Transgene Integration Site.

The MutaMouse transgenic rodent model is widely used for assessing in vivo mutagenicity. Here, we report the characterization of MutaMouse’s whole genome sequence and its genetic variants compared to the C57BL/6 reference genome. High coverage (>50X) next-generation sequencing (NGS) of whole genomes from multiple MutaMouse animals from the Health Canada (HC) colony showed ~5 million SNVs per genome, ~20% of which are putatively novel. Sequencing of two animals from a geographically separated colony at Covance indicated that, over the course of 23 years, each colony accumulated 47,847 (HC) and 17,677 (Covance) non-parental homozygous single nucleotide variants. We found no novel nonsense or missense mutations that impair the MutaMouse response to genotoxic agents. Pairing sequencing data with array comparative genomic hybridization (aCGH) improved the accuracy and resolution of copy number variants (CNVs) calls and identified 300 genomic regions with CNVs. We also used long-read sequence technology (PacBio) to show that the transgene integration site involved a large deletion event with multiple inversions and rearrangements near a retrotransposon. The MutaMouse genome gives important genetic context to studies using this model, offers insight on the mechanisms of structural variant formation, and contributes a framework to analyze aCGH results alongside NGS data.


April 21, 2020  |  

Genome analysis and genetic transformation of a water surface-floating microalga Chlorococcum sp. FFG039.

Microalgal harvesting and dewatering are the main bottlenecks that need to be overcome to tap the potential of microalgae for production of valuable compounds. Water surface-floating microalgae form robust biofilms, float on the water surface along with gas bubbles entrapped under the biofilms, and have great potential to overcome these bottlenecks. However, little is known about the molecular mechanisms involved in the water surface-floating phenotype. In the present study, we analysed the genome sequence of a water surface-floating microalga Chlorococcum sp. FFG039, with a next generation sequencing technique to elucidate the underlying mechanisms. Comparative genomics study with Chlorococcum sp. FFG039 and other non-floating green microalgae revealed some of the unique gene families belonging to this floating microalga, which may be involved in biofilm formation. Furthermore, genetic transformation of this microalga was achieved with an electroporation method. The genome information and transformation techniques presented in this study will be useful to obtain molecular insights into the water surface-floating phenotype of Chlorococcum sp. FFG039.


April 21, 2020  |  

Large Enriched Fragment Targeted Sequencing (LEFT-SEQ) Applied to Capture of Wolbachia Genomes.

Symbiosis is a major force of evolutionary change, influencing virtually all aspects of biology, from population ecology and evolution to genomics and molecular/biochemical mechanisms of development and reproduction. A remarkable example is Wolbachia endobacteria, present in some parasitic nematodes and many arthropod species. Acquisition of genomic data from diverse Wolbachia clades will aid in the elucidation of the different symbiotic mechanisms(s). However, challenges of de novo assembly of Wolbachia genomes include the presence in the sample of host DNA: nematode/vertebrate or insect. We designed biotinylated probes to capture large fragments of Wolbachia DNA for sequencing using PacBio technology (LEFT-SEQ: Large Enriched Fragment Targeted Sequencing). LEFT-SEQ was used to capture and sequence four Wolbachia genomes: the filarial nematode Brugia malayi, wBm, (21-fold enrichment), Drosophila mauritiana flies (2 isolates), wMau (11-fold enrichment), and Aedes albopictus mosquitoes, wAlbB (200-fold enrichment). LEFT-SEQ resulted in complete genomes for wBm and for wMau. For wBm, 18 single-nucleotide polymorphisms (SNPs), relative to the wBm reference, were identified and confirmed by PCR. A limit of LEFT-SEQ is illustrated by the wAlbB genome, characterized by a very high level of insertion sequences elements (ISs) and DNA repeats, for which only a 20-contig draft assembly was achieved.


April 21, 2020  |  

Extended insight into the Mycobacterium chelonae-abscessus complex through whole genome sequencing of Mycobacterium salmoniphilum outbreak and Mycobacterium salmoniphilum-like strains.

Members of the Mycobacterium chelonae-abscessus complex (MCAC) are close to the mycobacterial ancestor and includes both human, animal and fish pathogens. We present the genomes of 14 members of this complex: the complete genomes of Mycobacterium salmoniphilum and Mycobacterium chelonae type strains, seven M. salmoniphilum isolates, and five M. salmoniphilum-like strains including strains isolated during an outbreak in an animal facility at Uppsala University. Average nucleotide identity (ANI) analysis and core gene phylogeny revealed that the M. salmoniphilum-like strains are variants of the human pathogen Mycobacterium franklinii and phylogenetically close to Mycobacterium abscessus. Our data further suggested that M. salmoniphilum separates into three branches named group I, II and III with the M. salmoniphilum type strain belonging to group II. Among predicted virulence factors, the presence of phospholipase C (plcC), which is a major virulence factor that makes M. abscessus highly cytotoxic to mouse macrophages, and that M. franklinii originally was isolated from infected humans make it plausible that the outbreak in the animal facility was caused by a M. salmoniphilum-like strain. Interestingly, M. salmoniphilum-like was isolated from tap water suggesting that it can be present in the environment. Moreover, we predicted the presence of mutational hotspots in the M. salmoniphilum isolates and 26% of these hotspots overlap with genes categorized as having roles in virulence, disease and defense. We also provide data about key genes involved in transcription and translation such as sigma factor, ribosomal protein and tRNA genes.


April 21, 2020  |  

Long-read assembly of the Chinese rhesus macaque genome and identification of ape-specific structural variants.

We present a high-quality de novo genome assembly (rheMacS) of the Chinese rhesus macaque (Macaca mulatta) using long-read sequencing and multiplatform scaffolding approaches. Compared to the current Indian rhesus macaque reference genome (rheMac8), rheMacS increases sequence contiguity 75-fold, closing 21,940 of the remaining assembly gaps (60.8 Mbp). We improve gene annotation by generating more than two million full-length transcripts from ten different tissues by long-read RNA sequencing. We sequence resolve 53,916 structural variants (96% novel) and identify 17,000 ape-specific structural variants (ASSVs) based on comparison to ape genomes. Many ASSVs map within ChIP-seq predicted enhancer regions where apes and macaque show diverged enhancer activity and gene expression. We further characterize a subset that may contribute to ape- or great-ape-specific phenotypic traits, including taillessness, brain volume expansion, improved manual dexterity, and large body size. The rheMacS genome assembly serves as an ideal reference for future biomedical and evolutionary studies.


April 21, 2020  |  

Meiotic sex in Chagas disease parasite Trypanosoma cruzi.

Genetic exchange enables parasites to rapidly transform disease phenotypes and exploit new host populations. Trypanosoma cruzi, the parasitic agent of Chagas disease and a public health concern throughout Latin America, has for decades been presumed to exchange genetic material rarely and without classic meiotic sex. We present compelling evidence from 45 genomes sequenced from southern Ecuador that T. cruzi in fact maintains truly sexual, panmictic groups that can occur alongside others that remain highly clonal after past hybridization events. These groups with divergent reproductive strategies appear genetically isolated despite possible co-occurrence in vectors and hosts. We propose biological explanations for the fine-scale disconnectivity we observe and discuss the epidemiological consequences of flexible reproductive modes. Our study reinvigorates the hunt for the site of genetic exchange in the T. cruzi life cycle, provides tools to define the genetic determinants of parasite virulence, and reforms longstanding theory on clonality in trypanosomatid parasites.


April 21, 2020  |  

CRISPR/CAS9 targeted CAPTURE of mammalian genomic regions for characterization by NGS.

The robust detection of structural variants in mammalian genomes remains a challenge. It is particularly difficult in the case of genetically unstable Chinese hamster ovary (CHO) cell lines with only draft genome assemblies available. We explore the potential of the CRISPR/Cas9 system for the targeted capture of genomic loci containing integrated vectors in CHO-K1-based cell lines followed by next generation sequencing (NGS), and compare it to popular target-enrichment sequencing methods and to whole genome sequencing (WGS). Three different CRISPR/Cas9-based techniques were evaluated; all of them allow for amplification-free enrichment of target genomic regions in the range from 5 to 60 fold, and for recovery of ~15 kb-long sequences with no sequencing artifacts introduced. The utility of these protocols has been proven by the identification of transgene integration sites and flanking sequences in three CHO cell lines. The long enriched fragments helped to identify Escherichia coli genome sequences co-integrated with vectors, and were further characterized by Whole Genome Sequencing (WGS). Other advantages of CRISPR/Cas9-based methods are the ease of bioinformatics analysis, potential for multiplexing, and the production of long target templates for real-time sequencing.


April 21, 2020  |  

Genome analysis of the rice coral Montipora capitata.

Corals comprise a biomineralizing cnidarian, dinoflagellate algal symbionts, and associated microbiome of prokaryotes and viruses. Ongoing efforts to conserve coral reefs by identifying the major stress response pathways and thereby laying the foundation to select resistant genotypes rely on a robust genomic foundation. Here we generated and analyzed a high quality long-read based ~886 Mbp nuclear genome assembly and transcriptome data from the dominant rice coral, Montipora capitata from Hawai’i. Our work provides insights into the architecture of coral genomes and shows how they differ in size and gene inventory, putatively due to population size variation. We describe a recent example of foreign gene acquisition via a bacterial gene transfer agent and illustrate the major pathways of stress response that can be used to predict regulatory components of the transcriptional networks in M. capitata. These genomic resources provide insights into the adaptive potential of these sessile, long-lived species in both natural and human influenced environments and facilitate functional and population genomic studies aimed at Hawaiian reef restoration and conservation.


April 21, 2020  |  

Complete Genome Sequence of Sequevar 14M Ralstonia solanacearum Strain HA4-1 Reveals Novel Type III Effectors Acquired Through Horizontal Gene Transfer.

Ralstonia solanacearum, which causes bacterial wilt in a broad range of plants, is considered a “species complex” due to its significant genetic diversity. Recently, we have isolated a new R. solanacearum strain HA4-1 from Hong’an county in Hubei province of China and identified it being phylotype I, sequevar 14M (phylotype I-14M). Interestingly, we found that it can cause various disease symptoms among different potato genotypes and display different pathogenic behavior compared to a phylogenetically related strain, GMI1000. To dissect the pathogenic mechanisms of HA4-1, we sequenced its whole genome by combined sequencing technologies including Illumina HiSeq2000, PacBio RS II, and BAC-end sequencing. Genome assembly results revealed the presence of a conventional chromosome, a megaplasmid as well as a 143 kb plasmid in HA4-1. Comparative genome analysis between HA4-1 and GMI1000 shows high conservation of the general virulence factors such as secretion systems, motility, exopolysaccharides (EPS), and key regulatory factors, but significant variation in the repertoire and structure of type III effectors, which could be the determinants of their differential pathogenesis in certain potato species or genotypes. We have identified two novel type III effectors that were probably acquired through horizontal gene transfer (HGT). These novel R. solanacearum effectors display homology to several YopJ and XopAC family members. We named them as RipBR and RipBS. Notably, the copy of RipBR on the plasmid is a pseudogene, while the other on the megaplasmid is normal. For RipBS, there are three copies located in the megaplasmid and plasmid, respectively. Our results have not only enriched the genome information on R. solanacearum species complex by sequencing the first sequevar 14M strain and the largest plasmid reported in R. solanacearum to date but also revealed the variation in the repertoire of type III effectors. This will greatly contribute to the future studies on the pathogenic evolution, host adaptation, and interaction between R. solanacearum and potato.


April 21, 2020  |  

Genomic Analyses Reveal Evidence of Independent Evolution, Demographic History, and Extreme Environment Adaptation of Tibetan Plateau Agaricus bisporus.

Agaricus bisporus distributed in the Tibetan Plateau of China has high-stress resistance that is valuable for breeding improvements. However, its evolutionary history, specialization, and adaptation to the extreme Tibetan Plateau environment are largely unknown. Here, we performed de novo genome sequencing of a representative Tibetan Plateau wild strain ABM and comparative genomic analysis with the reported European strain H97 and H39. The assembled ABM genome was 30.4 Mb in size, and comprised 8,562 protein-coding genes. The ABM genome shared highly conserved syntenic blocks and a few inversions with H97 and H39. The phylogenetic tree constructed by 1,276 single-copy orthologous genes in nine fungal species showed that the Tibetan Plateau and European A. bisporus diverged ~5.5 million years ago. Population genomic analysis using genome resequencing of 29 strains revealed that the Tibetan Plateau population underwent significant differentiation from the European and American populations and evolved independently, and the global climate changes critically shaped the demographic history of the Tibetan Plateau population. Moreover, we identified key genes that are related to the cell wall and membrane system, and the development and defense systems regulated A. bisporus adapting to the harsh Tibetan Plateau environment. These findings highlight the value of genomic data in assessing the evolution and adaptation of mushrooms and will enhance future genetic improvements of A. bisporus.


April 21, 2020  |  

Comparative Genomics of Marine Sponge-Derived Streptomyces spp. Isolates SM17 and SM18 With Their Closest Terrestrial Relatives Provides Novel Insights Into Environmental Niche Adaptations and Secondary Metabolite Biosynthesis Potential.

The emergence of antibiotic resistant microorganisms has led to an increased need for the discovery and development of novel antimicrobial compounds. Frequent rediscovery of the same natural products (NPs) continues to decrease the likelihood of the discovery of new compounds from soil bacteria. Thus, efforts have shifted toward investigating microorganisms and their secondary metabolite biosynthesis potential, from diverse niche environments, such as those isolated from marine sponges. Here we investigated at the genomic level two Streptomyces spp. strains, namely SM17 and SM18, isolated from the marine sponge Haliclona simulans, with previously reported antimicrobial activity against clinically relevant pathogens; using single molecule real-time (SMRT) sequencing. We performed a series of comparative genomic analyses on SM17 and SM18 with their closest terrestrial relatives, namely S. albus J1074 and S. pratensis ATCC 33331 respectively; in an effort to provide further insights into potential environmental niche adaptations (ENAs) of marine sponge-associated Streptomyces, and on how these adaptations might be linked to their secondary metabolite biosynthesis potential. Prediction of secondary metabolite biosynthetic gene clusters (smBGCs) indicated that, even though the marine isolates are closely related to their terrestrial counterparts at a genomic level; they potentially produce different compounds. SM17 and SM18 displayed a better ability to grow in high salinity medium when compared to their terrestrial counterparts, and further analysis of their genomes indicated that they possess a pool of 29 potential ENA genes that are absent in S. albus J1074 and S. pratensis ATCC 33331. This ENA gene pool included functional categories of genes that are likely to be related to niche adaptations and which could be grouped based on potential biological functions such as osmotic stress, defense; transcriptional regulation; symbiotic interactions; antimicrobial compound production and resistance; ABC transporters; together with horizontal gene transfer and defense-related features.


April 21, 2020  |  

Mobilome of Brevibacterium aurantiacum Sheds Light on Its Genetic Diversity and Its Adaptation to Smear-Ripened Cheeses.

Brevibacterium aurantiacum is an actinobacterium that confers key organoleptic properties to washed-rind cheeses during the ripening process. Although this industrially relevant species has been gaining an increasing attention in the past years, its genome plasticity is still understudied due to the unavailability of complete genomic sequences. To add insights on the mobilome of this group, we sequenced the complete genomes of five dairy Brevibacterium strains and one non-dairy strain using PacBio RSII. We performed phylogenetic and pan-genome analyses, including comparisons with other publicly available Brevibacterium genomic sequences. Our phylogenetic analysis revealed that these five dairy strains, previously identified as Brevibacterium linens, belong instead to the B. aurantiacum species. A high number of transposases and integrases were observed in the Brevibacterium spp. strains. In addition, we identified 14 and 12 new insertion sequences (IS) in B. aurantiacum and B. linens genomes, respectively. Several stretches of homologous DNA sequences were also found between B. aurantiacum and other cheese rind actinobacteria, suggesting horizontal gene transfer (HGT). A HGT region from an iRon Uptake/Siderophore Transport Island (RUSTI) and an iron uptake composite transposon were found in five B. aurantiacum genomes. These findings suggest that low iron availability in milk is a driving force in the adaptation of this bacterial species to this niche. Moreover, the exchange of iron uptake systems suggests cooperative evolution between cheese rind actinobacteria. We also demonstrated that the integrative and conjugative element BreLI (Brevibacterium Lanthipeptide Island) can excise from B. aurantiacum SMQ-1417 chromosome. Our comparative genomic analysis suggests that mobile genetic elements played an important role into the adaptation of B. aurantiacum to cheese ecosystems.


April 21, 2020  |  

A reference-grade wild soybean genome.

Efficient crop improvement depends on the application of accurate genetic information contained in diverse germplasm resources. Here we report a reference-grade genome of wild soybean accession W05, with a final assembled genome size of 1013.2?Mb and a contig N50 of 3.3?Mb. The analytical power of the W05 genome is demonstrated by several examples. First, we identify an inversion at the locus determining seed coat color during domestication. Second, a translocation event between chromosomes 11 and 13 of some genotypes is shown to interfere with the assignment of QTLs. Third, we find a region containing copy number variations of the Kunitz trypsin inhibitor (KTI) genes. Such findings illustrate the power of this assembly in the analysis of large structural variations in soybean germplasm collections. The wild soybean genome assembly has wide applications in comparative genomic and evolutionary studies, as well as in crop breeding and improvement programs.


April 21, 2020  |  

Complete genome sequence analysis of the thermoacidophilic verrucomicrobial methanotroph “Candidatus Methylacidiphilum kamchatkense” strain Kam1 and comparison with its closest relatives.

The candidate genus “Methylacidiphilum” comprises thermoacidophilic aerobic methane oxidizers belonging to the Verrucomicrobia phylum. These are the first described non-proteobacterial aerobic methane oxidizers. The genes pmoCAB, encoding the particulate methane monooxygenase do not originate from horizontal gene transfer from proteobacteria. Instead, the “Ca. Methylacidiphilum” and the sister genus “Ca. Methylacidimicrobium” represent a novel and hitherto understudied evolutionary lineage of aerobic methane oxidizers. Obtaining and comparing the full genome sequences is an important step towards understanding the evolution and physiology of this novel group of organisms.Here we present the closed genome of “Ca. Methylacidiphilum kamchatkense” strain Kam1 and a comparison with the genomes of its two closest relatives “Ca. Methylacidiphilum fumariolicum” strain SolV and “Ca. Methylacidiphilum infernorum” strain V4. The genome consists of a single 2,2 Mbp chromosome with 2119 predicted protein coding sequences. Genome analysis showed that the majority of the genes connected with metabolic traits described for one member of “Ca. Methylacidiphilum” is conserved between all three genomes. All three strains encode class I CRISPR-cas systems. The average nucleotide identity between “Ca. M. kamchatkense” strain Kam1 and strains SolV and V4 is =95% showing that they should be regarded as separate species. Whole genome comparison revealed a high degree of synteny between the genomes of strains Kam1 and SolV. In contrast, comparison of the genomes of strains Kam1 and V4 revealed a number of rearrangements. There are large differences in the numbers of transposable elements found in the genomes of the three strains with 12, 37 and 80 transposable elements in the genomes of strains Kam1, V4 and SolV respectively. Genomic rearrangements and the activity of transposable elements explain much of the genomic differences between strains. For example, a type 1h uptake hydrogenase is conserved between strains Kam1 and SolV but seems to have been lost from strain V4 due to genomic rearrangements.Comparing three closed genomes of “Ca. Methylacidiphilum” spp. has given new insights into the evolution of these organisms and revealed large differences in numbers of transposable elements between strains, the activity of these explains much of the genomic differences between strains.


April 21, 2020  |  

A Phage-Like Plasmid Carrying blaKPC-2 Gene in Carbapenem-Resistant Pseudomonas aeruginosa.

Background: Lateral gene transfer plays a central role in the dissemination of carbapenem resistance in bacterial pathogens associated with nosocomial infections, mainly Enterobacteriaceae and Pseudomonas aeruginosa. Despite their clinical significance, there is little information regarding the mobile genetic elements and mechanism of acquisition and propagation of lateral genes in P. aeruginosa, and they remain largely unknown. Objectives: The present study characterized the genetic context of blaKPC-2 in carbapenem-resistant P. aeruginosa strain BH9. Methods:Pseudomonas aeruginosa BH9 sequencing was performed using the long-read PacBio SMRT platform and the Ion Proton System. De novo assembly was carried out using the SMRT pipeline and Canu, and gene prediction and annotation were performed using Prokka and RAST. Results:Pseudomonas aeruginosa BH9 exhibited a 7.1 Mb circular chromosome. However, the blaKPC-2 gene is located in an additional contig composed by a small plasmid pBH6 from P. aeruginosa strain BH6 and several phage-related genes. Further analysis revealed that the beginning and end of the contig contain identical sequences, supporting a circular plasmid structure. This structure spans 41,087 bp, exhibiting all the Mu-like phage landmarks. In addition, 5-bp direct repeats (GGATG) flanking the pBH6 ends were found, strongly indicating integration of the Mu-like phage into the pBH6 plasmid. Mu phages are commonly found in P. aeruginosa. However, for the first time showing a potential impact in shaping the vehicles of the dissemination of antimicrobial (e.g., plasmid pBH6) resistance genes in the Pseudomonas genus. Conclusion: pBH6 captured the Mu-like Phage BH9, creating a co-integrate pBH6::Phage BH9, and this phage-plasmid complex may represent novel case of a phage-like plasmid.


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