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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

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

Pentatricopeptide repeat poly(A) binding protein KPAF4 stabilizes mitochondrial mRNAs in Trypanosoma brucei.

In Trypanosoma brucei, most mitochondrial mRNAs undergo editing, and 3′ adenylation and uridylation. The internal sequence changes and terminal extensions are coordinated: pre-editing addition of the short (A) tail protects the edited transcript against 3′-5′ degradation, while post-editing A/U-tailing renders mRNA competent for translation. Participation of a poly(A) binding protein (PABP) in coupling of editing and 3′ modification processes has been inferred, but its identity and mechanism of action remained elusive. We report identification of KPAF4, a pentatricopeptide repeat-containing PABP which sequesters the A-tail and impedes mRNA degradation. Conversely, KPAF4 inhibits uridylation of A-tailed transcripts and, therefore, premature A/U-tailing of partially-edited mRNAs. This quality check point likely prevents translation of incompletely edited mRNAs. We also find that RNA editing substrate binding complex (RESC) mediates the interaction between the 5′ end-bound pyrophosphohydrolase MERS1 and 3′ end-associated KPAF4 to enable mRNA circularization. This event appears to be critical for edited mRNA stability.

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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

Complete genome sequence of 3-chlorobenzoate-degrading bacterium Cupriavidus necator NH9 and reclassification of the strains of the genera Cupriavidus and Ralstonia based on phylogenetic and whole-genome sequence analyses.

Cupriavidus necator NH9, a 3-chlorobenzoate (3-CB)-degrading bacterium, was isolated from soil in Japan. In this study, the complete genome sequence of NH9 was obtained via PacBio long-read sequencing to better understand the genetic components contributing to the strain’s ability to degrade aromatic compounds, including 3-CB. The genome of NH9 comprised two circular chromosomes (4.3 and 3.4 Mb) and two circular plasmids (427 and 77 kb) containing 7,290 coding sequences, 15 rRNA and 68 tRNA genes. Kyoto Encyclopedia of Genes and Genomes pathway analysis of the protein-coding sequences in NH9 revealed a capacity to completely degrade benzoate, 2-, 3-, or 4-hydroxybenzoate, 2,3-, 2,5-, or 3,4-dihydroxybenzoate, benzoylformate, and benzonitrile. To validate the identification of NH9, phylogenetic analyses (16S rRNA sequence-based tree and multilocus sequence analysis) and whole-genome sequence analyses (average nucleotide identity, percentage of conserved proteins, and tetra-nucleotide analyses) were performed, confirming that NH9 is a C. necator strain. Over the course of our investigation, we noticed inconsistencies in the classification of several strains that were supposed to belong to the two closely-related genera Cupriavidus and Ralstonia. As a result of whole-genome sequence analysis of 46 Cupriavidus strains and 104 Ralstonia strains, we propose that the taxonomic classification of 41 of the 150 strains should be changed. Our results provide a clear delineation of the two genera based on genome sequences, thus allowing taxonomic identification of strains belonging to these two genera.

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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

Valinomycin, produced by Streptomyces sp. S8, a key antifungal metabolite in large patch disease suppressiveness.

Large patch disease, caused by Rhizoctonia solani AG2-2, is the most devastating disease in Zoysiagrass (Zoysia japonica). Current large patch disease control strategies rely primarily upon the use of chemical pesticides. Streptomyces sp. S8 is known to possess exceptional antagonistic properties that could potentially suppress the large patch pathogen found at turfgrass plantations. This study aims to demonstrate the feasibility of using the strain as a biological control mechanism. Sequencing of the S8 strain genome revealed a valinomycin biosynthesis gene cluster. This cluster is composed of the vlm1 and vlm2 genes, which are known to produce antifungal compounds. In order to verify this finding for the large patch pathogen, a valinomycin biosynthesis knockout mutant was created via the CRISPR/Cas9 system. The mutant lost antifungal activity against the large patch pathogen. Consequently, it is anticipated that eco-friendly microbial preparations derived from the S8 strain can be utilized to biologically control large patch disease.

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April 21, 2020

New genetic context of lnu(B) composed of two multi-resistance gene clusters in clinical Streptococcus agalactiae ST-19 strains.

Clindamycin is a lincosamide antibiotic used to treat staphylococcal and streptococcal infections. Reports of clinical Streptococcus agalactiae isolates with the rare lincosamide resistance/macrolide susceptibility (LR/MS) phenotype are increasing worldwide. In this study, we characterised three clinical S. agalactiae strains with the unusual L phenotype from China.Three clinical S. agalactiae strains, Sag3, Sag27 and Sag4104, with the L phenotype were identified from 186 isolates collected from 2016 to 2018 in Shanghai, China. The MICs of clindamycin, erythromycin, tetracycline, levofloxacin, and penicillin were determined using Etest. PCR for the lnu(B) gene was conducted. Whole genome sequencing and sequence analysis were carried out to investigate the genetic context of lnu(B). Efforts to transfer lincomycin resistance by conjugation and to identify the circular form by inverse PCR were made.Sag3, Sag27, and Sag4104 were susceptible to erythromycin (MIC =0.25?mg/L) but resistant to clindamycin (MIC =1?mg/L). lnu(B) was found to be responsible for the L phenotype. lnu(B) in Sag3 and Sag27 were chromosomally located in an aadE-spw-lsa(E)-lnu(B) resistance gene cluster adjacent to an upstream 7-kb tet(L)-cat resistance gene cluster. Two resistance gene clusters were flanked by the IS6-like element, IS1216. Sag4104 only contained partial genes of aadE-spw-lsa(E)-lnu(B) resistance gene cluster and was also flanked by IS1216.These results established the presence of the L phenotype associated with lnu(B) in clinical S. agalactiae isolates in China. The lnu(B)-containing multi-resistance gene cluster possibly acts as a composite transposon flanked by IS1216 and as a vehicle for the dissemination of multidrug resistance among S. agalactiae.

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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

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

Transcriptomic profiles of 33 opium poppy samples in different tissues, growth phases, and cultivars.

Opium poppy is one of the most important medicinal plants and remains the only commercial resource of morphinan-based painkillers. However, little is known about the regulatory mechanisms involved in benzylisoquinoline alkaloids (BIAs) biosynthesis in opium poppy. Herein, the full-length transcriptome dataset of opium poppy was constructed for the first time in accompanied with the 33 samples of Illumina transcriptome data from different tissues, growth phases and cultivars. The long-read sequencing produced 902,140 raw reads with 55,114 high-quality transcripts, and short-read sequencing produced 1,923,679,864 clean reads with an average Q30 rate of 93%. The high-quality transcripts were subsequently quantified using the short reads, and the expression of each unigene among different samples was calculated as reads per kilobase per million mapped reads (RPKM). These data provide a foundation for opium poppy transcriptomic analysis, which may aid in capturing splice variants and some non-coding RNAs involved in the regulation of BIAs biosynthesis. It can also be used for genome assembly and annotation which will favor in new transcript identification.

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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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