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April 21, 2020

Genome Features and Secondary Metabolites Biosynthetic Potential of the Class Ktedonobacteria.

The prevalence of antibiotic resistance and the decrease in novel antibiotic discovery in recent years necessitates the identification of potentially novel microbial resources to produce natural products. Ktedonobacteria, a class of deeply branched bacterial lineage in the ancient phylum Chloroflexi, are ubiquitous in terrestrial environments and characterized by their large genome size and complex life cycle. These characteristics indicate Ktedonobacteria as a potential active producer of bioactive compounds. In this study, we observed the existence of a putative “megaplasmid,” multiple copies of ribosomal RNA operons, and high ratio of hypothetical proteins with unknown functions in the class Ktedonobacteria. Furthermore, a total of 104 antiSMASH-predicted putative biosynthetic gene clusters (BGCs) for secondary metabolites with high novelty and diversity were identified in nine Ktedonobacteria genomes. Our investigation of domain composition and organization of the non-ribosomal peptide synthetase and polyketide synthase BGCs further supports the concept that class Ktedonobacteria may produce compounds structurally different from known natural products. Furthermore, screening of bioactive compounds from representative Ktedonobacteria strains resulted in the identification of broad antimicrobial activities against both Gram-positive and Gram-negative tested bacterial strains. Based on these findings, we propose the ancient, ubiquitous, and spore-forming Ktedonobacteria as a versatile and promising microbial resource for natural product discovery.

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April 21, 2020

Sequence properties of certain GC rich avian genes, their origins and absence from genome assemblies: case studies.

More and more eukaryotic genomes are sequenced and assembled, most of them presented as a complete model in which missing chromosomal regions are filled by Ns and where a few chromosomes may be lacking. Avian genomes often contain sequences with high GC content, which has been hypothesized to be at the origin of many missing sequences in these genomes. We investigated features of these missing sequences to discover why some may not have been integrated into genomic libraries and/or sequenced.The sequences of five red jungle fowl cDNA models with high GC content were used as queries to search publicly available datasets of Illumina and Pacbio sequencing reads. These were used to reconstruct the leptin, TNFa, MRPL52, PCP2 and PET100 genes, all of which are absent from the red jungle fowl genome model. These gene sequences displayed elevated GC contents, had intron sizes that were sometimes larger than non-avian orthologues, and had non-coding regions that contained numerous tandem and inverted repeat sequences with motifs able to assemble into stable G-quadruplexes and intrastrand dyadic structures. Our results suggest that Illumina technology was unable to sequence the non-coding regions of these genes. On the other hand, PacBio technology was able to sequence these regions, but with dramatically lower efficiency than would typically be expected.High GC content was not the principal reason why numerous GC-rich regions of avian genomes are missing from genome assembly models. Instead, it is the presence of tandem repeats containing motifs capable of assembling into very stable secondary structures that is likely responsible.

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April 21, 2020

Prediction of Host-Specific Genes by Pan-Genome Analyses of the Korean Ralstonia solanacearum Species Complex.

The soil-borne pathogenic Ralstonia solanacearum species complex (RSSC) is a group of plant pathogens that is economically destructive worldwide and has a broad host range, including various solanaceae plants, banana, ginger, sesame, and clove. Previously, Korean RSSC strains isolated from samples of potato bacterial wilt were grouped into four pathotypes based on virulence tests against potato, tomato, eggplant, and pepper. In this study, we sequenced the genomes of 25 Korean RSSC strains selected based on these pathotypes. The newly sequenced genomes were analyzed to determine the phylogenetic relationships between the strains with average nucleotide identity values, and structurally compared via multiple genome alignment using Mauve software. To identify candidate genes responsible for the host specificity of the pathotypes, functional genome comparisons were conducted by analyzing pan-genome orthologous group (POG) and type III secretion system effectors (T3es). POG analyses revealed that a total of 128 genes were shared only in tomato-non-pathogenic strains, 8 genes in tomato-pathogenic strains, 5 genes in eggplant-non-pathogenic strains, 7 genes in eggplant-pathogenic strains, 1 gene in pepper-non-pathogenic strains, and 34 genes in pepper-pathogenic strains. When we analyzed T3es, three host-specific effectors were predicted: RipS3 (SKWP3) and RipH3 (HLK3) were found only in tomato-pathogenic strains, and RipAC (PopC) were found only in eggplant-pathogenic strains. Overall, we identified host-specific genes and effectors that may be responsible for virulence functions in RSSC in silico. The expected characters of those genes suggest that the host range of RSSC is determined by the comprehensive actions of various virulence factors, including effectors, secretion systems, and metabolic enzymes.

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April 21, 2020

Closing the Yield Gap for Cannabis: A Meta-Analysis of Factors Determining Cannabis Yield.

Until recently, the commercial production of Cannabis sativa was restricted to varieties that yielded high-quality fiber while producing low levels of the psychoactive cannabinoid tetrahydrocannabinol (THC). In the last few years, a number of jurisdictions have legalized the production of medical and/or recreational cannabis with higher levels of THC, and other jurisdictions seem poised to follow suit. Consequently, demand for industrial-scale production of high yield cannabis with consistent cannabinoid profiles is expected to increase. In this paper we highlight that currently, projected annual production of cannabis is based largely on facility size, not yield per square meter. This meta-analysis of cannabis yields reported in scientific literature aimed to identify the main factors contributing to cannabis yield per plant, per square meter, and per W of lighting electricity. In line with previous research we found that variety, plant density, light intensity and fertilization influence cannabis yield and cannabinoid content; we also identified pot size, light type and duration of the flowering period as predictors of yield and THC accumulation. We provide insight into the critical role of light intensity, quality, and photoperiod in determining cannabis yields, with particular focus on the potential for light-emitting diodes (LEDs) to improve growth and reduce energy requirements. We propose that the vast amount of genomics data currently available for cannabis can be used to better understand the effect of genotype on yield. Finally, we describe diversification that is likely to emerge in cannabis growing systems and examine the potential role of plant-growth promoting rhizobacteria (PGPR) for growth promotion, regulation of cannabinoid biosynthesis, and biocontrol.

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April 21, 2020

Divergent evolutionary trajectories following speciation in two ectoparasitic honey bee mites.

Multispecies host-parasite evolution is common, but how parasites evolve after speciating remains poorly understood. Shared evolutionary history and physiology may propel species along similar evolutionary trajectories whereas pursuing different strategies can reduce competition. We test these scenarios in the economically important association between honey bees and ectoparasitic mites by sequencing the genomes of the sister mite species Varroa destructor and Varroa jacobsoni. These genomes were closely related, with 99.7% sequence identity. Among the 9,628 orthologous genes, 4.8% showed signs of positive selection in at least one species. Divergent selective trajectories were discovered in conserved chemosensory gene families (IGR, SNMP), and Halloween genes (CYP) involved in moulting and reproduction. However, there was little overlap in these gene sets and associated GO terms, indicating different selective regimes operating on each of the parasites. Based on our findings, we suggest that species-specific strategies may be needed to combat evolving parasite communities. © The Author(s) 2019.

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April 21, 2020

The sequencing and de novo assembly of the Larimichthys crocea genome using PacBio and Hi-C technologies.

Larimichthys crocea is an endemic marine fish in East Asia that belongs to Sciaenidae in Perciformes. L. crocea has now been recognized as an “iconic” marine fish species in China because not only is it a popular food fish in China, it is a representative victim of overfishing and still provides high value fish products supported by the modern large-scale mariculture industry. Here, we report a chromosome-level reference genome of L. crocea generated by employing the PacBio single molecule sequencing technique (SMRT) and high-throughput chromosome conformation capture (Hi-C) technologies. The genome sequences were assembled into 1,591 contigs with a total length of 723.86?Mb and a contig N50 length of 2.83?Mb. After chromosome-level scaffolding, 24 scaffolds were constructed with a total length of 668.67?Mb (92.48% of the total length). Genome annotation identified 23,657 protein-coding genes and 7262 ncRNAs. This highly accurate, chromosome-level reference genome of L. crocea provides an essential genome resource to support the development of genome-scale selective breeding and restocking strategies of L. crocea.

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April 21, 2020

The sequence and de novo assembly of Takifugu bimaculatus genome using PacBio and Hi-C technologies.

Takifugu bimaculatus is a native teleost species of the southeast coast of China where it has been cultivated as an important edible fish in the last decade. Genetic breeding programs, which have been recently initiated for improving the aquaculture performance of T. bimaculatus, urgently require a high-quality reference genome to facilitate genome selection and related genetic studies. To address this need, we produced a chromosome-level reference genome of T. bimaculatus using the PacBio single molecule sequencing technique (SMRT) and High-through chromosome conformation capture (Hi-C) technologies. The genome was assembled into 2,193 contigs with a total length of 404.21?Mb and a contig N50 length of 1.31?Mb. After chromosome-level scaffolding, 22 chromosomes with a total length of 371.68?Mb were constructed. Moreover, a total of 21,117 protein-coding genes and 3,471 ncRNAs were annotated in the reference genome. The highly accurate, chromosome-level reference genome of T. bimaculatus provides an essential genome resource for not only the genome-scale selective breeding of T. bimaculatus but also the exploration of the evolutionary basis of the speciation and local adaptation of the Takifugu genus.

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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

One reference genome is not enough

A recent study on human structural variation indicates insufficiencies and errors in the human reference genome, GRCh38, and argues for the construction of a human pan-genome.

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April 21, 2020

A high-quality de novo genome assembly from a single mosquito using PacBio sequencing

A high-quality reference genome is a fundamental resource for functional genetics, comparative genomics, and population genomics, and is increasingly important for conservation biology. PacBio Single Molecule, Real-Time (SMRT) sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. Improvements in throughput and concomitant reductions in cost have made PacBio an attractive core technology for many large genome initiatives, however, relatively high DNA input requirements (~5 µg for standard library protocol) have placed PacBio out of reach for many projects on small organisms that have lower DNA content, or on projects with limited input DNA for other reasons. Here we present a high-quality de novo genome assembly from a single Anopheles coluzzii mosquito. A modified SMRTbell library construction protocol without DNA shearing and size selection was used to generate a SMRTbell library from just 100 ng of starting genomic DNA. The sample was run on the Sequel System with chemistry 3.0 and software v6.0, generating, on average, 25 Gb of sequence per SMRT Cell with 20 h movies, followed by diploid de novo genome assembly with FALCON-Unzip. The resulting curated assembly had high contiguity (contig N50 3.5 Mb) and completeness (more than 98% of conserved genes were present and full-length). In addition, this single-insect assembly now places 667 (>90%) of formerly unplaced genes into their appropriate chromosomal contexts in the AgamP4 PEST reference. We were also able to resolve maternal and paternal haplotypes for over 1/3 of the genome. By sequencing and assembling material from a single diploid individual, only two haplotypes were present, simplifying the assembly process compared to samples from multiple pooled individuals. The method presented here can be applied to samples with starting DNA amounts as low as 100 ng per 1 Gb genome size. This new low-input approach puts PacBio-based assemblies in reach for small highly heterozygous organisms that comprise much of the diversity of life.

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April 21, 2020

Construction of JRG (Japanese reference genome) with single-molecule real-time sequencing

In recent genome analyses, population-specific reference panels have indicated important. However, reference panels based on short-read sequencing data do not sufficiently cover long insertions. Therefore, the nature of long insertions has not been well documented. Here, we assembled a Japanese genome using single-molecule real-time sequencing data and characterized insertions found in the assembled genome. We identified 3691 insertions ranging from 100?bps to ~10,000?bps in the assembled genome relative to the international reference sequence (GRCh38). To validate and characterize these insertions, we mapped short-reads from 1070 Japanese individuals and 728 individuals from eight other populations to insertions integrated into GRCh38. With this result, we constructed JRGv1 (Japanese Reference Genome version 1) by integrating the 903 verified insertions, totaling 1,086,173 bases, shared by at least two Japanese individuals into GRCh38. We also constructed decoyJRGv1 by concatenating 3559 verified insertions, totaling 2,536,870 bases, shared by at least two Japanese individuals or by six other assemblies. This assembly improved the alignment ratio by 0.4% on average. These results demonstrate the importance of refining the reference assembly and creating a population-specific reference genome. JRGv1 and decoyJRGv1 are available at the JRG website.

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October 23, 2019

Simultaneous non-contiguous deletions using large synthetic DNA and site-specific recombinases.

Toward achieving rapid and large scale genome modification directly in a target organism, we have developed a new genome engineering strategy that uses a combination of bioinformatics aided design, large synthetic DNA and site-specific recombinases. Using Cre recombinase we swapped a target 126-kb segment of the Escherichia coli genome with a 72-kb synthetic DNA cassette, thereby effectively eliminating over 54 kb of genomic DNA from three non-contiguous regions in a single recombination event. We observed complete replacement of the native sequence with the modified synthetic sequence through the action of the Cre recombinase and no competition from homologous recombination. Because of the versatility and high-efficiency of the Cre-lox system, this method can be used in any organism where this system is functional as well as adapted to use with other highly precise genome engineering systems. Compared to present-day iterative approaches in genome engineering, we anticipate this method will greatly speed up the creation of reduced, modularized and optimized genomes through the integration of deletion analyses data, transcriptomics, synthetic biology and site-specific recombination. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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October 23, 2019

A high quality assembly of the Nile Tilapia (Oreochromis niloticus) genome reveals the structure of two sex determination regions.

Tilapias are the second most farmed fishes in the world and a sustainable source of food. Like many other fish, tilapias are sexually dimorphic and sex is a commercially important trait in these fish. In this study, we developed a significantly improved assembly of the tilapia genome using the latest genome sequencing methods and show how it improves the characterization of two sex determination regions in two tilapia species.A homozygous clonal XX female Nile tilapia (Oreochromis niloticus) was sequenced to 44X coverage using Pacific Biosciences (PacBio) SMRT sequencing. Dozens of candidate de novo assemblies were generated and an optimal assembly (contig NG50 of 3.3Mbp) was selected using principal component analysis of likelihood scores calculated from several paired-end sequencing libraries. Comparison of the new assembly to the previous O. niloticus genome assembly reveals that recently duplicated portions of the genome are now well represented. The overall number of genes in the new assembly increased by 27.3%, including a 67% increase in pseudogenes. The new tilapia genome assembly correctly represents two recent vasa gene duplication events that have been verified with BAC sequencing. At total of 146Mbp of additional transposable element sequence are now assembled, a large proportion of which are recent insertions. Large centromeric satellite repeats are assembled and annotated in cichlid fish for the first time. Finally, the new assembly identifies the long-range structure of both a ~9Mbp XY sex determination region on LG1 in O. niloticus, and a ~50Mbp WZ sex determination region on LG3 in the related species O. aureus.This study highlights the use of long read sequencing to correctly assemble recent duplications and to characterize repeat-filled regions of the genome. The study serves as an example of the need for high quality genome assemblies and provides a framework for identifying sex determining genes in tilapia and related fish species.

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October 23, 2019

Accurate identification and quantification of DNA species by next-generation sequencing in adeno-associated viral vectors produced in insect cells.

Recombinant adeno-associated viral (rAAV) vectors have proven excellent tools for the treatment of many genetic diseases and other complex diseases. However, the illegitimate encapsidation of DNA contaminants within viral particles constitutes a major safety concern for rAAV-based therapies. Moreover, the development of rAAV vectors for early-phase clinical trials has revealed the limited accuracy of the analytical tools used to characterize these new and complex drugs. Although most published data concerning residual DNA in rAAV preparations have been generated by quantitative PCR, we have developed a novel single-strand virus sequencing (SSV-Seq) method for quantification of DNA contaminants in AAV vectors produced in mammalian cells by next-generation sequencing (NGS). Here, we describe the adaptation of SSV-Seq for the accurate identification and quantification of DNA species in rAAV stocks produced in insect cells. We found that baculoviral DNA was the most abundant contaminant, representing less than 2.1% of NGS reads regardless of serotype (2, 8, or rh10). Sf9 producer cell DNA was detected at low frequency (=0.03%) in rAAV lots. Advanced computational analyses revealed that (1) baculoviral sequences close to the inverted terminal repeats preferentially underwent illegitimate encapsidation, and (2) single-nucleotide variants were absent from the rAAV genome. The high-throughput sequencing protocol described here enables effective DNA quality control of rAAV vectors produced in insect cells, and is adapted to conform with regulatory agency safety requirements.

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