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September 22, 2019  |  

Assembly of chromosome-scale contigs by efficiently resolving repetitive sequences with long reads

Due to the large number of repetitive sequences in complex eukaryotic genomes, fragmented and incompletely assembled genomes lose value as reference sequences, often due to short contigs that cannot be anchored or mispositioned onto chromosomes. Here we report a novel method Highly Efficient Repeat Assembly (HERA), which includes a new concept called a connection graph as well as algorithms for constructing the graph. HERA resolves repeats at high efficiency with single-molecule sequencing data, and enables the assembly of chromosome-scale contigs by further integrating genome maps and Hi-C data. We tested HERA with the genomes of rice R498, maize B73, human HX1 and Tartary buckwheat Pinku1. HERA can correctly assemble most of the tandemly repetitive sequences in rice using single-molecule sequencing data only. Using the same maize and human sequencing data published by Jiao et al. (2017) and Shi et al. (2016), respectively, we dramatically improved on the sequence contiguity compared with the published assemblies, increasing the contig N50 from 1.3 Mb to 61.2 Mb in maize B73 assembly and from 8.3 Mb to 54.4 Mb in human HX1 assembly with HERA. We provided a high-quality maize reference genome with 96.9% of the gaps filled (only 76 gaps left) and several incorrectly positioned sequences fixed compared with the B73 RefGen_v4 assembly. Comparisons between the HERA assembly of HX1 and the human GRCh38 reference genome showed that many gaps in GRCh38 could be filled, and that GRCh38 contained some potential errors that could be fixed. We assembled the Pinku1 genome into 12 scaffolds with a contig N50 size of 27.85 Mb. HERA serves as a new genome assembly/phasing method to generate high quality sequences for complex genomes and as a curation tool to improve the contiguity and completeness of existing reference genomes, including the correction of assembly errors in repetitive regions.

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September 22, 2019  |  

Genome Assembly.

Genome assembly uses sequence similarity to go from sequencing reads to longer contiguous sequences (contigs). Scaffolds are contigs linked together by gaps where the order and orientation of the contigs is known but the exact sequence connecting two contigs is unknown, represented by Ns which estimate the gap length. Here we describe recommendations for genome assembly for different sequencing technologies, describe organelle assembly, and review how to perform assembly quality control.

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September 22, 2019  |  

Improved de novo genome assembly and analysis of the Chinese cucurbit Siraitia grosvenorii, also known as monk fruit or luo-han-guo.

Luo-han-guo (Siraitia grosvenorii), also called monk fruit, is a member of the Cucurbitaceae family. Monk fruit has become an important area for research because of the pharmacological and economic potential of its noncaloric, extremely sweet components (mogrosides). It is also commonly used in traditional Chinese medicine for the treatment of lung congestion, sore throat, and constipation. Recently, a single reference genome became available for monk fruit, assembled from 36.9x genome coverage reads via Illumina sequencing platforms. This genome assembly has a relatively short (34.2 kb) contig N50 length and lacks integrated annotations. These drawbacks make it difficult to use as a reference in assembling transcriptomes and discovering novel functional genes.Here, we offer a new high-quality draft of the S. grosvenorii genome assembled using 31 Gb (~73.8x) long single molecule real time sequencing reads and polished with ~50 Gb Illumina paired-end reads. The final genome assembly is approximately 469.5 Mb, with a contig N50 length of 432,384 bp, representing a 12.6-fold improvement. We further annotated 237.3 Mb of repetitive sequence and 30,565 consensus protein coding genes with combined evidence. Phylogenetic analysis showed that S. grosvenorii diverged from members of the Cucurbitaceae family approximately 40.9 million years ago. With comprehensive transcriptomic analysis and differential expression testing, we identified 4,606 up-regulated genes in the early fruit compared to the leaf, a number of which were linked to metabolic pathways regulating fruit development and ripening.The availability of this new monk fruit genome assembly, as well as the annotations, will facilitate the discovery of new functional genes and the genetic improvement of monk fruit.

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September 22, 2019  |  

Three substrains of the cyanobacterium Anabaena sp. PCC 7120 display divergence in genomic sequences and hetC function.

Anabaena sp. strain PCC 7120 is a model strain for molecular studies of cell differentiation and patterning in heterocyst-forming cyanobacteria. Subtle differences in heterocyst development have been noticed in different laboratories working on the same organism. In this study, 360 mutations, including single nucleotide polymorphisms (SNPs), small insertion/deletions (indels; 1 to 3 bp), fragment deletions, and transpositions, were identified in the genomes of three substrains. Heterogeneous/heterozygous bases were also identified due to the polyploidy nature of the genome and the multicellular morphology but could be completely segregated when plated after filament fragmentation by sonication. hetC is a gene upregulated in developing cells during heterocyst formation in Anabaena sp. strain PCC 7120 and found in approximately half of other heterocyst-forming cyanobacteria. Inactivation of hetC in 3 substrains of Anabaena sp. PCC 7120 led to different phenotypes: the formation of heterocysts, differentiating cells that keep dividing, or the presence of both heterocysts and dividing differentiating cells. The expression of P hetZ -gfp in these hetC mutants also showed different patterns of green fluorescent protein (GFP) fluorescence. Thus, the function of hetC is influenced by the genomic background and epistasis and constitutes an example of evolution under way.IMPORTANCE Our knowledge about the molecular genetics of heterocyst formation, an important cell differentiation process for global N2 fixation, is mostly based on studies with Anabaena sp. strain PCC 7120. Here, we show that rapid microevolution is under way in this strain, leading to phenotypic variations for certain genes related to heterocyst development, such as hetC This study provides an example for ongoing microevolution, marked by multiple heterogeneous/heterozygous single nucleotide polymorphisms (SNPs), in a multicellular multicopy-genome microorganism. Copyright © 2018 American Society for Microbiology.

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September 22, 2019  |  

De novo assembly of a young Drosophila Y chromosome using single-molecule sequencing and chromatin conformation capture.

While short-read sequencing technology has resulted in a sharp increase in the number of species with genome assemblies, these assemblies are typically highly fragmented. Repeats pose the largest challenge for reference genome assembly, and pericentromeric regions and the repeat-rich Y chromosome are typically ignored from sequencing projects. Here, we assemble the genome of Drosophila miranda using long reads for contig formation, chromatin interaction maps for scaffolding and short reads, and optical mapping and bacterial artificial chromosome (BAC) clone sequencing for consensus validation. Our assembly recovers entire chromosomes and contains large fractions of repetitive DNA, including about 41.5 Mb of pericentromeric and telomeric regions, and >100 Mb of the recently formed highly repetitive neo-Y chromosome. While Y chromosome evolution is typically characterized by global sequence loss and shrinkage, the neo-Y increased in size by almost 3-fold because of the accumulation of repetitive sequences. Our high-quality assembly allows us to reconstruct the chromosomal events that have led to the unusual sex chromosome karyotype in D. miranda, including the independent de novo formation of a pair of sex chromosomes at two distinct time points, or the reversion of a former Y chromosome to an autosome.

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September 22, 2019  |  

Stendomycin and pantomycin are identical natural products: Preparation of a functionalized bioactive analogue.

The natural products pantomycin and stendomycin were both reported as antimicrobial agents. We demonstrate by gene cluster analysis, LC-MS analysis, and isolation that these polypeptides are identical, and we identify previously unknown congeners. We show that stendomycin can be chemically modified at its electrophilic dehydrobutyrine moiety yielding the first bioactive analogue of this natural product which can undergo additional functionalization. This compound may be a valuable starting point for molecular probe development, and we invite its distribution to the scientific community.

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September 22, 2019  |  

High-quality assembly of the reference genome for scarlet sage, Salvia splendens, an economically important ornamental plant.

Salvia splendens Ker-Gawler, scarlet or tropical sage, is a tender herbaceous perennial widely introduced and seen in public gardens all over the world. With few molecular resources, breeding is still restricted to traditional phenotypic selection, and the genetic mechanisms underlying phenotypic variation remain unknown. Hence, a high-quality reference genome will be very valuable for marker-assisted breeding, genome editing, and molecular genetics.We generated 66 Gb and 37 Gb of raw DNA sequences, respectively, from whole-genome sequencing of a largely homozygous scarlet sage inbred line using Pacific Biosciences (PacBio) single-molecule real-time and Illumina HiSeq sequencing platforms. The PacBio de novo assembly yielded a final genome with a scaffold N50 size of 3.12 Mb and a total length of 808 Mb. The repetitive sequences identified accounted for 57.52% of the genome sequence, and ?54,008 protein-coding genes were predicted collectively with ab initio and homology-based gene prediction from the masked genome. The divergence time between S. splendens and Salvia miltiorrhiza was estimated at 28.21 million years ago (Mya). Moreover, 3,797 species-specific genes and 1,187 expanded gene families were identified for the scarlet sage genome.We provide the first genome sequence and gene annotation for the scarlet sage. The availability of these resources will be of great importance for further breeding strategies, genome editing, and comparative genomics among related species.

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September 22, 2019  |  

A graph-based approach to diploid genome assembly.

Constructing high-quality haplotype-resolved de novo assemblies of diploid genomes is important for revealing the full extent of structural variation and its role in health and disease. Current assembly approaches often collapse the two sequences into one haploid consensus sequence and, therefore, fail to capture the diploid nature of the organism under study. Thus, building an assembler capable of producing accurate and complete diploid assemblies, while being resource-efficient with respect to sequencing costs, is a key challenge to be addressed by the bioinformatics community.We present a novel graph-based approach to diploid assembly, which combines accurate Illumina data and long-read Pacific Biosciences (PacBio) data. We demonstrate the effectiveness of our method on a pseudo-diploid yeast genome and show that we require as little as 50× coverage Illumina data and 10× PacBio data to generate accurate and complete assemblies. Additionally, we show that our approach has the ability to detect and phase structural variants.https://github.com/whatshap/whatshap.Supplementary data are available at Bioinformatics online.

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September 22, 2019  |  

Whole genome and transcriptome maps of the entirely black native Korean chicken breed Yeonsan Ogye.

Yeonsan Ogye (YO), an indigenous Korean chicken breed (Gallus gallus domesticus), has entirely black external features and internal organs. In this study, the draft genome of YO was assembled using a hybrid de novo assembly method that takes advantage of high-depth Illumina short reads (376.6X) and low-depth Pacific Biosciences (PacBio) long reads (9.7X).The contig and scaffold NG50s of the hybrid de novo assembly were 362.3 Kbp and 16.8 Mbp, respectively. The completeness (97.6%) of the draft genome (Ogye_1.1) was evaluated with single-copy orthologous genes using Benchmarking Universal Single-Copy Orthologs and found to be comparable to the current chicken reference genome (galGal5; 97.4%; contigs were assembled with high-depth PacBio long reads (50X) and scaffolded with short reads) and superior to other avian genomes (92%-93%; assembled with short read-only or hybrid methods). Compared to galGal4 and galGal5, the draft genome included 551 structural variations including the fibromelanosis (FM) locus duplication, related to hyperpigmentation. To comprehensively reconstruct transcriptome maps, RNA sequencing and reduced representation bisulfite sequencing data were analyzed from 20 tissues, including 4 black tissues (skin, shank, comb, and fascia). The maps included 15,766 protein-coding and 6,900 long noncoding RNA genes, many of which were tissue-specifically expressed and displayed tissue-specific DNA methylation patterns in the promoter regions.We expect that the resulting genome sequence and transcriptome maps will be valuable resources for studying domestic chicken breeds, including black-skinned chickens, as well as for understanding genomic differences between breeds and the evolution of hyperpigmented chickens and functional elements related to hyperpigmentation.

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September 22, 2019  |  

Genome analysis of the ancient tracheophyte Selaginella tamariscina reveals evolutionary features relevant to the acquisition of desiccation tolerance.

Resurrection plants, which are the “gifts” of natural evolution, are ideal models for studying the genetic basis of plant desiccation tolerance. Here, we report a high-quality genome assembly of 301 Mb for the diploid spike moss Selaginella tamariscina, a primitive vascular resurrection plant. We predicated 27 761 protein-coding genes from the assembled S. tamariscina genome, 11.38% (2363) of which showed significant expression changes in response to desiccation. Approximately 60.58% of the S. tamariscina genome was annotated as repetitive DNA, which is an almost 2-fold increase of that in the genome of desiccation-sensitive Selaginella moellendorffii. Genomic and transcriptomic analyses highlight the unique evolution and complex regulations of the desiccation response in S. tamariscina, including species-specific expansion of the oleosin and pentatricopeptide repeat gene families, unique genes and pathways for reactive oxygen species generation and scavenging, and enhanced abscisic acid (ABA) biosynthesis and potentially distinct regulation of ABA signaling and response. Comparative analysis of chloroplast genomes of several Selaginella species revealed a unique structural rearrangement and the complete loss of chloroplast NAD(P)H dehydrogenase (NDH) genes in S. tamariscina, suggesting a link between the absence of the NDH complex and desiccation tolerance. Taken together, our comparative genomic and transcriptomic analyses reveal common and species-specific desiccation tolerance strategies in S. tamariscina, providing significant insights into the desiccation tolerance mechanism and the evolution of resurrection plants. Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.

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September 22, 2019  |  

Potential survival and pathogenesis of a novel strain, Vibrio parahaemolyticus FORC_022, isolated from a soy sauce marinated crab by genome and transcriptome analyses.

Vibrio parahaemolyticus can cause gastrointestinal illness through consumption of seafood. Despite frequent food-borne outbreaks of V. parahaemolyticus, only 19 strains have subjected to complete whole-genome analysis. In this study, a novel strain of V. parahaemolyticus, designated FORC_022 (Food-borne pathogen Omics Research Center_022), was isolated from soy sauce marinated crabs, and its genome and transcriptome were analyzed to elucidate the pathogenic mechanisms. FORC_022 did not include major virulence factors of thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). However, FORC_022 showed high cytotoxicity and had several V. parahaemolyticus islands (VPaIs) and other virulence factors, such as various secretion systems (types I, II, III, IV, and VI), in comparative genome analysis with CDC_K4557 (the most similar strain) and RIMD2210633 (genome island marker strain). FORC_022 harbored additional virulence genes, including accessory cholera enterotoxin, zona occludens toxin, and tight adhesion (tad) locus, compared with CDC_K4557. In addition, O3 serotype specific gene and the marker gene of pandemic O3:K6 serotype (toxRS) were detected in FORC_022. The expressions levels of genes involved in adherence and carbohydrate transporter were high, whereas those of genes involved in motility, arginine biosynthesis, and proline metabolism were low after exposure to crabs. Moreover, the virulence factors of the type III secretion system, tad locus, and thermolabile hemolysin were overexpressed. Therefore, the risk of foodborne-illness may be high following consumption of FORC_022 contaminated crab. These results provided molecular information regarding the survival and pathogenesis of V. parahaemolyticus FORC_022 strain in contaminated crab and may have applications in food safety.

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September 22, 2019  |  

Biology and genome of a newly discovered sibling species of Caenorhabditis elegans.

A ‘sibling’ species of the model organism Caenorhabditis elegans has long been sought for use in comparative analyses that would enable deep evolutionary interpretations of biological phenomena. Here, we describe the first sibling species of C. elegans, C. inopinata n. sp., isolated from fig syconia in Okinawa, Japan. We investigate the morphology, developmental processes and behaviour of C. inopinata, which differ significantly from those of C. elegans. The 123-Mb C. inopinata genome was sequenced and assembled into six nuclear chromosomes, allowing delineation of Caenorhabditis genome evolution and revealing unique characteristics, such as highly expanded transposable elements that might have contributed to the genome evolution of C. inopinata. In addition, C. inopinata exhibits massive gene losses in chemoreceptor gene families, which could be correlated with its limited habitat area. We have developed genetic and molecular techniques for C. inopinata; thus C. inopinata provides an exciting new platform for comparative evolutionary studies.

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September 22, 2019  |  

Genomic analysis of the insect-killing fungus Beauveria bassiana JEF-007 as a biopesticide.

Insect-killing fungi have high potential in pest management. A deeper insight into the fungal genes at the whole genome level is necessary to understand the inter-species or intra-species genetic diversity of fungal genes, and to select excellent isolates. In this work, we conducted a whole genome sequencing of Beauveria bassiana (Bb) JEF-007 and characterized pathogenesis-related features and compared with other isolates including Bb ARSEF2860. A large number of Bb JEF-007 genes showed high identity with Bb ARSEF2860, but some genes showed moderate or low identity. The two Bb isolates showed a significant difference in vegetative growth, antibiotic-susceptibility, and virulence against Tenebrio molitor larvae. When highly identical genes between the two Bb isolates were subjected to real-time PCR, their transcription levels were different, particularly in heat shock protein 30 (hsp30) gene which is related to conidial thermotolerance. In several B. bassiana isolates, chitinases and trypsin-like protease genes involved in pathogenesis were highly conserved, but other genes showed noticeable sequence variation within the same species. Given the transcriptional and genetic diversity in B. bassiana, a selection of virulent isolates with industrial advantages is a pre-requisite, and this genetic approach could support the development of excellent biopesticides with intellectual property protection.

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September 22, 2019  |  

Opposite polarity monospore genome de novo sequencing and comparative analysis reveal the possible heterothallic life cycle of Morchella importuna.

Morchella is a popular edible fungus worldwide due to its rich nutrition and unique flavor. Many research efforts were made on the domestication and cultivation of Morchella all over the world. In recent years, the cultivation of Morchella was successfully commercialized in China. However, the biology is not well understood, which restricts the further development of the morel fungus cultivation industry. In this paper, we performed de novo sequencing and assembly of the genomes of two monospores with a different mating type (M04M24 and M04M26) isolated from the commercially cultivated strain M04. Gene annotation and comparative genome analysis were performed to study differences in CAZyme (Carbohydrate-active enzyme) enzyme content, transcription factors, duplicated sequences, structure of mating type sites, and differences at the gene and functional levels between the two monospore strains of M. importuna. Results showed that the de novo assembled haploid M04M24 and M04M26 genomes were 48.98 and 51.07 Mb, respectively. A complete fine physical map of M. importuna was obtained from genome coverage and gene completeness evaluation. A total of 10,852 and 10,902 common genes and 667 and 868 endemic genes were identified from the two monospore strains, respectively. The Gene Ontology (GO) and KAAS (KEGG Automatic Annotation Serve) enrichment analyses showed that the endemic genes performed different functions. The two monospore strains had 99.22% collinearity with each other, accompanied with certain position and rearrangement events. Analysis of complete mating-type loci revealed that the two monospore M. importuna strains contained an independent mating-type structure and remained conserved in sequence and location. The phylogenetic and divergence time of M. importuna was analyzed at the whole-genome level for the first time. The bifurcation time of morel and tuber was estimated to be 201.14 million years ago (Mya); the two monospore strains with a different mating type represented the evolution of different nuclei, and the single copy homologous genes between them were also different due to a genetic differentiation distance about 0.65 Mya. Compared with truffles, M. importuna had an extension of 28 clusters of orthologous genes (COGs) and a contraction of two COGs. The two different polar nuclei with different degrees of contraction and expansion suggested that they might have undergone different evolutionary processes. The different mating-type structures, together with the functional clustering and enrichment analysis results of the endemic genes of the two different polar nuclei, imply that M. importuna might be a heterothallic fungus and the interaction between the endemic genes may be necessary for its complete life history. Studies on the genome of M. importuna facilitate a better understanding of morel biology and evolution.

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September 22, 2019  |  

The chromosome-level genome assemblies of two rattans (Calamus simplicifolius and Daemonorops jenkinsiana).

Calamus simplicifolius and Daemonorops jenkinsiana are two representative rattans, the most significant material sources for the rattan industry. However, the lack of reference genome sequences is a major obstacle for basic and applied biology on rattan.We produced two chromosome-level genome assemblies of C. simplicifolius and D. jenkinsiana using Illumina, Pacific Biosciences, and Hi-C sequencing data. A total of ~730 Gb and ~682 Gb of raw data covered the predicted genome lengths (~1.98 Gb of C. simplicifolius and ~1.61 Gb of D. jenkinsiana) to ~372 × and ~426 × read depths, respectively. The two de novo genome assemblies, ~1.94 Gb and ~1.58 Gb, were generated with scaffold N50s of ~160 Mb and ~119 Mb in C. simplicifolius and D. jenkinsiana, respectively. The C. simplicifolius and D. jenkinsiana genomes were predicted to harbor ?51,235 and ?53,342 intact protein-coding gene models, respectively. Benchmarking Universal Single-Copy Orthologs evaluation demonstrated that genome completeness reached 96.4% and 91.3% in the C. simplicifolius and D. jenkinsiana genomes, respectively. Genome evolution showed that four Arecaceae plants clustered together, and the divergence time between the two rattans was ~19.3 million years ago. Additionally, we identified 193 and 172 genes involved in the lignin biosynthesis pathway in the C. simplicifolius and D. jenkinsiana genomes, respectively.We present the first de novo assemblies of two rattan genomes (C. simplicifolius and D. jenkinsiana). These data will not only provide a fundamental resource for functional genomics, particularly in promoting germplasm utilization for breeding, but also serve as reference genomes for comparative studies between and among different species.

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