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April 21, 2020

A First Study of the Virulence Potential of a Bacillus subtilis Isolate From Deep-Sea Hydrothermal Vent.

Bacillus subtilis is the best studied Gram-positive bacterium, primarily as a model of cell differentiation and industrial exploitation. To date, little is known about the virulence of B. subtilis. In this study, we examined the virulence potential of a B. subtilis strain (G7) isolated from the Iheya North hydrothermal field of Okinawa Trough. G7 is aerobic, motile, endospore-forming, and requires NaCl for growth. The genome of G7 is composed of one circular chromosome of 4,216,133 base pairs with an average GC content of 43.72%. G7 contains 4,416 coding genes, 27.5% of which could not be annotated, and the remaining 72.5% were annotated with known or predicted functions in 25 different COG categories. Ten sets of 23S, 5S, and 16S ribosomal RNA operons, 86 tRNA and 14 sRNA genes, 50 tandem repeats, 41 mini-satellites, one microsatellite, and 42 transposons were identified in G7. Comparing to the genome of the B. subtilis wild type strain NCIB 3610T, G7 genome contains many genomic translocations, inversions, and insertions, and twice the amount of genomic Islands (GIs), with 42.5% of GI genes encoding hypothetical proteins. G7 possesses abundant putative virulence genes associated with adhesion, invasion, dissemination, anti-phagocytosis, and intracellular survival. Experimental studies showed that G7 was able to cause mortality in fish and mice following intramuscular/intraperitoneal injection, resist the killing effect of serum complement, and replicate in mouse macrophages and fish peripheral blood leukocytes. Taken together, our study indicates that G7 is a B. subtilis isolate with unique genetic features and can be lethal to vertebrate animals once being introduced into the animals by artificial means. These results provide the first insight into the potential harmfulness of deep-sea B. subtilis.

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April 21, 2020

Metaepigenomic analysis reveals the unexplored diversity of DNA methylation in an environmental prokaryotic community.

DNA methylation plays important roles in prokaryotes, and their genomic landscapes-prokaryotic epigenomes-have recently begun to be disclosed. However, our knowledge of prokaryotic methylation systems is focused on those of culturable microbes, which are rare in nature. Here, we used single-molecule real-time and circular consensus sequencing techniques to reveal the ‘metaepigenomes’ of a microbial community in the largest lake in Japan, Lake Biwa. We reconstructed 19 draft genomes from diverse bacterial and archaeal groups, most of which are yet to be cultured. The analysis of DNA chemical modifications in those genomes revealed 22 methylated motifs, nine of which were novel. We identified methyltransferase genes likely responsible for methylation of the novel motifs, and confirmed the catalytic specificities of four of them via transformation experiments using synthetic genes. Our study highlights metaepigenomics as a powerful approach for identification of the vast unexplored variety of prokaryotic DNA methylation systems in nature.

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April 21, 2020

Assignment of virus and antimicrobial resistance genes to microbial hosts in a complex microbial community by combined long-read assembly and proximity ligation.

We describe a method that adds long-read sequencing to a mix of technologies used to assemble a highly complex cattle rumen microbial community, and provide a comparison to short read-based methods. Long-read alignments and Hi-C linkage between contigs support the identification of 188 novel virus-host associations and the determination of phage life cycle states in the rumen microbial community. The long-read assembly also identifies 94 antimicrobial resistance genes, compared to only seven alleles in the short-read assembly. We demonstrate novel techniques that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.

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April 21, 2020

Long-read based de novo assembly of low-complexity metagenome samples results in finished genomes and reveals insights into strain diversity and an active phage system.

Complete and contiguous genome assemblies greatly improve the quality of subsequent systems-wide functional profiling studies and the ability to gain novel biological insights. While a de novo genome assembly of an isolated bacterial strain is in most cases straightforward, more informative data about co-existing bacteria as well as synergistic and antagonistic effects can be obtained from a direct analysis of microbial communities. However, the complexity of metagenomic samples represents a major challenge. While third generation sequencing technologies have been suggested to enable finished metagenome-assembled genomes, to our knowledge, the complete genome assembly of all dominant strains in a microbiome sample has not been demonstrated. Natural whey starter cultures (NWCs) are used in cheese production and represent low-complexity microbiomes. Previous studies of Swiss Gruyère and selected Italian hard cheeses, mostly based on amplicon metagenomics, concurred that three species generally pre-dominate: Streptococcus thermophilus, Lactobacillus helveticus and Lactobacillus delbrueckii.Two NWCs from Swiss Gruyère producers were subjected to whole metagenome shotgun sequencing using the Pacific Biosciences Sequel and Illumina MiSeq platforms. In addition, longer Oxford Nanopore Technologies MinION reads had to be generated for one to resolve repeat regions. Thereby, we achieved the complete assembly of all dominant bacterial genomes from these low-complexity NWCs, which was corroborated by a 16S rRNA amplicon survey. Moreover, two distinct L. helveticus strains were successfully co-assembled from the same sample. Besides bacterial chromosomes, we could also assemble several bacterial plasmids and phages and a corresponding prophage. Biologically relevant insights were uncovered by linking the plasmids and phages to their respective host genomes using DNA methylation motifs on the plasmids and by matching prokaryotic CRISPR spacers with the corresponding protospacers on the phages. These results could only be achieved by employing long-read sequencing data able to span intragenomic as well as intergenomic repeats.Here, we demonstrate the feasibility of complete de novo genome assembly of all dominant strains from low-complexity NWCs based on whole metagenomics shotgun sequencing data. This allowed to gain novel biological insights and is a fundamental basis for subsequent systems-wide omics analyses, functional profiling and phenotype to genotype analysis of specific microbial communities.

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April 21, 2020

Cichorium intybus L.?×?Cicerbita alpina Walbr.: doubled haploid chicory induction and CENH3 characterization

Intergeneric hybridization between industrial chicory (Cichorium intybus L.) and Cicerbita alpina Walbr. induces interspecific hybrids and haploid chicory plants after in vitro embryo rescue. The protocol yielded haploids in 5 out of 12 cultivars pollinated; altogether 18 haploids were regenerated from 2836 embryos, with a maximum efficiency of 1.96% haploids per cross. Obtained haploids were chromosome doubled with mitosis inhibitors trifluralin and oryzalin; exposure to 0.05 g L-1 oryzalin during one week was the most efficient treatment to regenerate doubled haploids. Inbreeding effects in vitro were limited, but the ploidy level affects morphology. Transcriptome sequencing revealed two unique copies of CENH3 in Cicerbita alpina Walbr. Comparison of CENH3.1 protein sequences of Cicerbita and Cichorium obtained through transcriptome and whole shotgun genome sequencing revealed two amino-acid substitutions at critical residues of the histone fold domain. These particular changes cause chromosome elimination and reduced centromere loading in several other species and might indicate a CENH3-dependent mechanism causing chromosome elimination of parental chromosomes during Cichorium?×?Cicerbita intergeneric hybridization. Our results provide insights in chromosome elimination and might increase the efficiency of haploid induction in Cichorium.

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April 21, 2020

Chromosome assembly of Collichthys lucidus, a fish of Sciaenidae with a multiple sex chromosome system.

Collichthys lucidus (C. lucidus) is a commercially important marine fish species distributed in coastal regions of East Asia with the X1X1X2X2/X1X2Y multiple sex chromosome system. The karyotype for female C. lucidus is 2n?=?48, while 2n?=?47 for male ones. Therefore, C. lucidus is also an excellent model to investigate teleost sex-determination and sex chromosome evolution. We reported the first chromosome genome assembly of C. lucidus using Illumina short-read, PacBio long-read sequencing and Hi-C technology. An 877?Mb genome was obtained with a contig and scaffold N50 of 1.1?Mb and 35.9?Mb, respectively. More than 97% BUSCOs genes were identified in the C. lucidus genome and 28,602 genes were annotated. We identified potential sex-determination genes along chromosomes and found that the chromosome 1 might be involved in the formation of Y specific metacentric chromosome. The first C. lucidus chromosome-level reference genome lays a solid foundation for the following population genetics study, functional gene mapping of important economic traits, sex-determination and sex chromosome evolution studies for Sciaenidae and teleosts.

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April 21, 2020

The wild sweetpotato (Ipomoea trifida) genome provides insights into storage root development.

Sweetpotato (Ipomoea batatas (L.) Lam.) is the seventh most important crop in the world and is mainly cultivated for its underground storage root (SR). The genetic studies of this species have been hindered by a lack of high-quality reference sequence due to its complex genome structure. Diploid Ipomoea trifida is the closest relative and putative progenitor of sweetpotato, which is considered a model species for sweetpotato, including genetic, cytological, and physiological analyses.Here, we generated the chromosome-scale genome sequence of SR-forming diploid I. trifida var. Y22 with high heterozygosity (2.20%). Although the chromosome-based synteny analysis revealed that the I. trifida shared conserved karyotype with Ipomoea nil after the separation, I. trifida had a much smaller genome than I. nil due to more efficient eliminations of LTR-retrotransposons and lack of species-specific amplification bursts of LTR-RTs. A comparison with four non-SR-forming species showed that the evolution of the beta-amylase gene family may be related to SR formation. We further investigated the relationship of the key gene BMY11 (with identity 47.12% to beta-amylase 1) with this important agronomic trait by both gene expression profiling and quantitative trait locus (QTL) mapping. And combining SR morphology and structure, gene expression profiling and qPCR results, we deduced that the products of the activity of BMY11 in splitting starch granules and be recycled to synthesize larger granules, contributing to starch accumulation and SR swelling. Moreover, we found the expression pattern of BMY11, sporamin proteins and the key genes involved in carbohydrate metabolism and stele lignification were similar to that of sweetpotato during the SR development.We constructed the high-quality genome reference of the highly heterozygous I. trifida through a combined approach and this genome enables a better resolution of the genomics feature and genome evolutions of this species. Sweetpotato SR development genes can be identified in I. trifida and these genes perform similar functions and patterns, showed that the diploid I. trifida var. Y22 with typical SR could be considered an ideal model for the studies of sweetpotato SR development.

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April 21, 2020

Characterization of a male specific region containing a candidate sex determining gene in Atlantic cod.

The genetic mechanisms determining sex in teleost fishes are highly variable and the master sex determining gene has only been identified in few species. Here we characterize a male-specific region of 9?kb on linkage group 11 in Atlantic cod (Gadus morhua) harboring a single gene named zkY for zinc knuckle on the Y chromosome. Diagnostic PCR test of phenotypically sexed males and females confirm the sex-specific nature of the Y-sequence. We identified twelve highly similar autosomal gene copies of zkY, of which eight code for proteins containing the zinc knuckle motif. 3D modeling suggests that the amino acid changes observed in six copies might influence the putative RNA-binding specificity. Cod zkY and the autosomal proteins zk1 and zk2 possess an identical zinc knuckle structure, but only the Y-specific gene zkY was expressed at high levels in the developing larvae before the onset of sex differentiation. Collectively these data suggest zkY as a candidate master masculinization gene in Atlantic cod. PCR amplification of Y-sequences in Arctic cod (Arctogadus glacialis) and Greenland cod (Gadus macrocephalus ogac) suggests that the male-specific region emerged in codfishes more than 7.5 million years ago.

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April 21, 2020

Haplotype-aware diplotyping from noisy long reads.

Current genotyping approaches for single-nucleotide variations rely on short, accurate reads from second-generation sequencing devices. Presently, third-generation sequencing platforms are rapidly becoming more widespread, yet approaches for leveraging their long but error-prone reads for genotyping are lacking. Here, we introduce a novel statistical framework for the joint inference of haplotypes and genotypes from noisy long reads, which we term diplotyping. Our technique takes full advantage of linkage information provided by long reads. We validate hundreds of thousands of candidate variants that have not yet been included in the high-confidence reference set of the Genome-in-a-Bottle effort.

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April 21, 2020

Comparative Phylogenomics, a Stepping Stone for Bird Biodiversity Studies

Birds are a group with immense availability of genomic resources, and hundreds of forthcoming genomes at the doorstep. We review recent developments in whole genome sequencing, phylogenomics, and comparative genomics of birds. Short read based genome assemblies are common, largely due to efforts of the Bird 10K genome project (B10K). Chromosome-level assemblies are expected to increase due to improved long-read sequencing. The available genomic data has enabled the reconstruction of the bird tree of life with increasing confidence and resolution, but challenges remain in the early splits of Neoaves due to their explosive diversification after the Cretaceous-Paleogene (K-Pg) event. Continued genomic sampling of the bird tree of life will not just better reflect their evolutionary history but also shine new light onto the organization of phylogenetic signal and conflict across the genome. The comparatively simple architecture of avian genomes makes them a powerful system to study the molecular foundation of bird specific traits. Birds are on the verge of becoming an extremely resourceful system to study biodiversity from the nucleotide up.

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April 21, 2020

A draft genome for Spatholobus suberectus.

Spatholobus suberectus Dunn (S. suberectus), which belongs to the Leguminosae, is an important medicinal plant in China. Owing to its long growth cycle and increased use in human medicine, wild resources of S. suberectus have decreased rapidly and may be on the verge of extinction. De novo assembly of the whole S. suberectus genome provides us a critical potential resource towards biosynthesis of the main bioactive components and seed development regulation mechanism of this plant. Utilizing several sequencing technologies such as Illumina HiSeq X Ten, single-molecule real-time sequencing, 10x Genomics, as well as new assembly techniques such as FALCON and chromatin interaction mapping (Hi-C), we assembled a chromosome-scale genome about 798?Mb in size. In total, 748?Mb (93.73%) of the contig sequences were anchored onto nine chromosomes with the longest scaffold being 103.57?Mb. Further annotation analyses predicted 31,634 protein-coding genes, of which 93.9% have been functionally annotated. All data generated in this study is available in public databases.

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April 21, 2020

Systematic analysis of dark and camouflaged genes reveals disease-relevant genes hiding in plain sight.

The human genome contains “dark” gene regions that cannot be adequately assembled or aligned using standard short-read sequencing technologies, preventing researchers from identifying mutations within these gene regions that may be relevant to human disease. Here, we identify regions with few mappable reads that we call dark by depth, and others that have ambiguous alignment, called camouflaged. We assess how well long-read or linked-read technologies resolve these regions.Based on standard whole-genome Illumina sequencing data, we identify 36,794 dark regions in 6054 gene bodies from pathways important to human health, development, and reproduction. Of these gene bodies, 8.7% are completely dark and 35.2% are =?5% dark. We identify dark regions that are present in protein-coding exons across 748 genes. Linked-read or long-read sequencing technologies from 10x Genomics, PacBio, and Oxford Nanopore Technologies reduce dark protein-coding regions to approximately 50.5%, 35.6%, and 9.6%, respectively. We present an algorithm to resolve most camouflaged regions and apply it to the Alzheimer’s Disease Sequencing Project. We rescue a rare ten-nucleotide frameshift deletion in CR1, a top Alzheimer’s disease gene, found in disease cases but not in controls.While we could not formally assess the association of the CR1 frameshift mutation with Alzheimer’s disease due to insufficient sample-size, we believe it merits investigating in a larger cohort. There remain thousands of potentially important genomic regions overlooked by short-read sequencing that are largely resolved by long-read technologies.

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April 21, 2020

High-coverage, long-read sequencing of Han Chinese trio reference samples.

Single-molecule long-read sequencing datasets were generated for a son-father-mother trio of Han Chinese descent that is part of the Genome in a Bottle (GIAB) consortium portfolio. The dataset was generated using the Pacific Biosciences Sequel System. The son and each parent were sequenced to an average coverage of 60 and 30, respectively, with N50 subread lengths between 16 and 18?kb. Raw reads and reads aligned to both the GRCh37 and GRCh38 are available at the NCBI GIAB ftp site (ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/ChineseTrio/). The GRCh38 aligned read data are archived in NCBI SRA (SRX4739017, SRX4739121, and SRX4739122). This dataset is available for anyone to develop and evaluate long-read bioinformatics methods.

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April 21, 2020

Experimental validation of in silico predicted RAD locus frequencies using genomic resources and short read data from a model marine mammal.

Restriction site-associated DNA sequencing (RADseq) has revolutionized the study of wild organisms by allowing cost-effective genotyping of thousands of loci. However, for species lacking reference genomes, it can be challenging to select the restriction enzyme that offers the best balance between the number of obtained RAD loci and depth of coverage, which is crucial for a successful outcome. To address this issue, PredRAD was recently developed, which uses probabilistic models to predict restriction site frequencies from a transcriptome assembly or other sequence resource based on either GC content or mono-, di- or trinucleotide composition. This program generates predictions that are broadly consistent with estimates of the true number of restriction sites obtained through in silico digestion of available reference genome assemblies. However, in practice the actual number of loci obtained could potentially differ as incomplete enzymatic digestion or patchy sequence coverage across the genome might lead to some loci not being represented in a RAD dataset, while erroneous assembly could potentially inflate the number of loci. To investigate this, we used genome and transcriptome assemblies together with RADseq data from the Antarctic fur seal (Arctocephalus gazella) to compare PredRAD predictions with empirical estimates of the number of loci obtained via in silico digestion and from de novo assemblies.PredRAD yielded consistently higher predicted numbers of restriction sites for the transcriptome assembly relative to the genome assembly. The trinucleotide and dinucleotide models also predicted higher frequencies than the mononucleotide or GC content models. Overall, the dinucleotide and trinucleotide models applied to the transcriptome and the genome assemblies respectively generated predictions that were closest to the number of restriction sites estimated by in silico digestion. Furthermore, the number of de novo assembled RAD loci mapping to restriction sites was similar to the expectation based on in silico digestion.Our study reveals generally high concordance between PredRAD predictions and empirical estimates of the number of RAD loci. This further supports the utility of PredRAD, while also suggesting that it may be feasible to sequence and assemble the majority of RAD loci present in an organism’s genome.

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April 21, 2020

In the name of the rose: a roadmap for rose research in the genome era.

The recent completion of the rose genome sequence is not the end of a process, but rather a starting point that opens up a whole set of new and exciting activities. Next to a high-quality genome sequence other genomic tools have also become available for rose, including transcriptomics data, a high-density single-nucleotide polymorphism array and software to perform linkage and quantitative trait locus mapping in polyploids. Rose cultivars are highly heterogeneous and diverse. This vast diversity in cultivated roses can be explained through the genetic potential of the genus, introgressions from wild species into commercial tetraploid germplasm and the inimitable efforts of historical breeders. We can now investigate how this diversity can best be exploited and refined in future breeding work, given the rich molecular toolbox now available to the rose breeding community. This paper presents possible lines of research now that rose has entered the genomics era, and attempts to partially answer the question that arises after the completion of any draft genome sequence: ‘Now that we have “the” genome, what’s next?’. Having access to a genome sequence will allow both (fundamental) scientific and (applied) breeding-orientated questions to be addressed. We outline possible approaches for a number of these questions.

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