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April 21, 2020

High-Quality Complete Genome Sequences of Three Pseudomonas aeruginosa Isolates Retrieved from Patients Hospitalized in Intensive Care Units.

Pseudomonas aeruginosa is one of the major Gram-negative pathogens responsible for hospital-acquired infections. Here, we present high-quality genome sequences of isolates from three P. aeruginosa genotypes retrieved from patients hospitalized in intensive care units. PacBio reads were assembled into a single contig, which was afterward corrected using Illumina HiSeq reads.

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April 21, 2020

Reconstruction of the genomes of drug-resistant pathogens for outbreak investigation through metagenomic sequencing

Culture-independent methods that target genome fragments have shown promise in identifying certain pathogens, but the holy grail of comprehensive pathogen genome detection from microbiologically complex samples for subsequent forensic analyses remains a challenge. In the context of an investigation of a nosocomial outbreak, we used shotgun metagenomic sequencing of a human fecal sample and a neural network algorithm based on tetranucleotide frequency profiling to reconstruct microbial genomes and tested the same approach using rectal swabs from a second patient. The approach rapidly and readily detected the genome of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae in the patient fecal specimen and in the rectal swab sample, achieving a level of strain resolution that was sufficient for confident transmission inference during a highly clonal outbreak. The analysis also detected previously unrecognized colonization of the patient by vancomycin-resistant Enterococcus faecium, another multidrug-resistant bacterium.IMPORTANCE The study results reported here perfectly demonstrate the power and promise of clinical metagenomics to recover genome sequences of important drug-resistant bacteria and to rapidly provide rich data that inform outbreak investigations and treatment decisions, independently of the need to culture the organisms.

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April 21, 2020

Complete Genome Sequence of “Candidatus Thioglobus sp.” Strain NP1, an Open-Ocean Isolate from the SUP05 Clade of Marine Gammaproteobacteria

Candidatus Thioglobus sp.textquotedblright strain NP1 is an open-ocean isolate from the SUP05 clade of Gammaproteobacteria. Whole-genome comparisons of strain NP1 to other sequenced isolates from the SUP05 clade indicate that it represents a new species of SUP05 that lacks the ability to fix inorganic carbon using the Calvin-Benson-Bassham cycle.

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April 21, 2020

An improved genome assembly of the fluke Schistosoma japonicum.

Schistosoma japonicum is a parasitic flatworm that causes human schistosomiasis, which is a significant cause of morbidity in China and the Philippines. A single draft genome was available for S. japonicum, yet this assembly is very fragmented and only covers 90% of the genome, which make it difficult to be applied as a reference in functional genome analysis and genes discovery.In this study, we present a high-quality assembly of the fluke S. japonicum genome by combining 20 G (~53X) long single molecule real time sequencing reads with 80 G (~ 213X) Illumina paired-end reads. This improved genome assembly is approximately 370.5 Mb, with contig and scaffold N50 length of 871.9 kb and 1.09 Mb, representing 142.4-fold and 6.2-fold improvement over the released WGS-based assembly, respectively. Additionally, our assembly captured 85.2% complete and 4.6% partial eukaryotic Benchmarking Universal Single-Copy Orthologs. Repetitive elements account for 46.80% of the genome, and 10,089 of the protein-coding genes were predicted from the improved genome, of which 96.5% have been functionally annotated. Lastly, using the improved assembly, we identified 20 significantly expanded gene families in S. japonicum, and those genes were primarily enriched in functions of proteolysis and protein glycosylation.Using the combination of PacBio and Illumina Sequencing technologies, we provided an improved high-quality genome of S. japonicum. This improved genome assembly, as well as the annotation, will be useful for the comparative genomics of the flukes and more importantly facilitate the molecular studies of this important parasite in the future.

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April 21, 2020

Parallels between natural selection in the cold-adapted crop-wild relative Tripsacum dactyloides and artificial selection in temperate adapted maize.

Artificial selection has produced varieties of domesticated maize that thrive in temperate climates around the world. However, the direct progenitor of maize, teosinte, is indigenous only to a relatively small range of tropical and subtropical latitudes and grows poorly or not at all outside of this region. Tripsacum, a sister genus to maize and teosinte, is naturally endemic to the majority of areas in the western hemisphere where maize is cultivated. A full-length reference transcriptome for Tripsacum dactyloides generated using long-read Iso-Seq data was used to characterize independent adaptation to temperate climates in this clade. Genes related to phospholipid biosynthesis, a critical component of cold acclimation in other cold-adapted plant lineages, were enriched among those genes experiencing more rapid rates of protein sequence evolution in T. dactyloides. In contrast with previous studies of parallel selection, we find that there is a significant overlap between the genes that were targets of artificial selection during the adaptation of maize to temperate climates and those that were targets of natural selection in temperate-adapted T. dactyloides. Genes related to growth, development, response to stimulus, signaling, and organelles were enriched in the set of genes identified as both targets of natural and artificial selection. © 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.

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April 21, 2020

A High-Quality Grapevine Downy Mildew Genome Assembly Reveals Rapidly Evolving and Lineage-Specific Putative Host Adaptation Genes.

Downy mildews are obligate biotrophic oomycete pathogens that cause devastating plant diseases on economically important crops. Plasmopara viticola is the causal agent of grapevine downy mildew, a major disease in vineyards worldwide. We sequenced the genome of Pl. viticola with PacBio long reads and obtained a new 92.94?Mb assembly with high contiguity (359 scaffolds for a N50 of 706.5?kb) due to a better resolution of repeat regions. This assembly presented a high level of gene completeness, recovering 1,592 genes encoding secreted proteins involved in plant-pathogen interactions. Plasmopara viticola had a two-speed genome architecture, with secreted protein-encoding genes preferentially located in gene-sparse, repeat-rich regions and evolving rapidly, as indicated by pairwise dN/dS values. We also used short reads to assemble the genome of Plasmopara muralis, a closely related species infecting grape ivy (Parthenocissus tricuspidata). The lineage-specific proteins identified by comparative genomics analysis included a large proportion of RxLR cytoplasmic effectors and, more generally, genes with high dN/dS values. We identified 270 candidate genes under positive selection, including several genes encoding transporters and components of the RNA machinery potentially involved in host specialization. Finally, the Pl. viticola genome assembly generated here will allow the development of robust population genomics approaches for investigating the mechanisms involved in adaptation to biotic and abiotic selective pressures in this species. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020

Human contamination in bacterial genomes has created thousands of spurious proteins.

Contaminant sequences that appear in published genomes can cause numerous problems for downstream analyses, particularly for evolutionary studies and metagenomics projects. Our large-scale scan of complete and draft bacterial and archaeal genomes in the NCBI RefSeq database reveals that 2250 genomes are contaminated by human sequence. The contaminant sequences derive primarily from high-copy human repeat regions, which themselves are not adequately represented in the current human reference genome, GRCh38. The absence of the sequences from the human assembly offers a likely explanation for their presence in bacterial assemblies. In some cases, the contaminating contigs have been erroneously annotated as containing protein-coding sequences, which over time have propagated to create spurious protein “families” across multiple prokaryotic and eukaryotic genomes. As a result, 3437 spurious protein entries are currently present in the widely used nr and TrEMBL protein databases. We report here an extensive list of contaminant sequences in bacterial genome assemblies and the proteins associated with them. We found that nearly all contaminants occurred in small contigs in draft genomes, which suggests that filtering out small contigs from draft genome assemblies may mitigate the issue of contamination while still keeping nearly all of the genuine genomic sequences. © 2019 Breitwieser et al.; Published by Cold Spring Harbor Laboratory Press.

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April 21, 2020

The Draft Genome of an Octocoral, Dendronephthya gigantea.

Coral reefs composed of stony corals are threatened by global marine environmental changes. However, soft coral communities of octocorallian species, appear more resilient. The genomes of several cnidarians species have been published, including from stony corals, sea anemones, and hydra. To fill the phylogenetic gap for octocoral species of cnidarians, we sequenced the octocoral, Dendronephthya gigantea, a nonsymbiotic soft coral, commonly known as the carnation coral. The D. gigantea genome size is ~276?Mb. A high-quality genome assembly was constructed from PacBio long reads (29.85 Gb with 108× coverage) and Illumina short paired-end reads (35.54 Gb with 128× coverage) resulting in the highest N50 value (1.4?Mb) reported thus far among cnidarian genomes. About 12% of the genome is repetitive elements and contained 28,879 predicted protein-coding genes. This gene set is composed of 94% complete BUSCO ortholog benchmark genes, which is the second highest value among the cnidarians, indicating high quality. Based on molecular phylogenetic analysis, octocoral and hexacoral divergence times were estimated at 544 MYA. There is a clear difference in Hox gene composition between these species: unlike hexacorals, the Antp superclass Evx gene was absent in D. gigantea. Here, we present the first genome assembly of a nonsymbiotic octocoral, D. gigantea to aid in the comparative genomic analysis of cnidarians, including stony and soft corals, both symbiotic and nonsymbiotic. The D. gigantea genome may also provide clues to mechanisms of differential coping between the soft and stony corals in response to scenarios of global warming. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020

The Reference Genome Sequence of Scutellaria baicalensis Provides Insights into the Evolution of Wogonin Biosynthesis.

Scutellaria baicalensis Georgi is important in Chinese traditional medicine where preparations of dried roots, “Huang Qin,” are used for liver and lung complaints and as complementary cancer treatments. We report a high-quality reference genome sequence for S. baicalensis where 93% of the 408.14-Mb genome has been assembled into nine pseudochromosomes with a super-N50 of 33.2 Mb. Comparison of this sequence with those of closely related species in the order Lamiales, Sesamum indicum and Salvia splendens, revealed that a specialized metabolic pathway for the synthesis of 4′-deoxyflavone bioactives evolved in the genus Scutellaria. We found that the gene encoding a specific cinnamate coenzyme A ligase likely obtained its new function following recent mutations, and that four genes encoding enzymes in the 4′-deoxyflavone pathway are present as tandem repeats in the genome of S. baicalensis. Further analyses revealed that gene duplications, segmental duplication, gene amplification, and point mutations coupled to gene neo- and subfunctionalizations were involved in the evolution of 4′-deoxyflavone synthesis in the genus Scutellaria. Our study not only provides significant insight into the evolution of specific flavone biosynthetic pathways in the mint family, Lamiaceae, but also will facilitate the development of tools for enhancing bioactive productivity by metabolic engineering in microbes or by molecular breeding in plants. The reference genome of S. baicalensis is also useful for improving the genome assemblies for other members of the mint family and offers an important foundation for decoding the synthetic pathways of bioactive compounds in medicinal plants.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.

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April 21, 2020

Long-read sequence and assembly of segmental duplications.

We have developed a computational method based on polyploid phasing of long sequence reads to resolve collapsed regions of segmental duplications within genome assemblies. Segmental Duplication Assembler (SDA; https://github.com/mvollger/SDA ) constructs graphs in which paralogous sequence variants define the nodes and long-read sequences provide attraction and repulsion edges, enabling the partition and assembly of long reads corresponding to distinct paralogs. We apply it to single-molecule, real-time sequence data from three human genomes and recover 33-79 megabase pairs (Mb) of duplications in which approximately half of the loci are diverged (<99.8%) compared to the reference genome. We show that the corresponding sequence is highly accurate (>99.9%) and that the diverged sequence corresponds to copy-number-variable paralogs that are absent from the human reference genome. Our method can be applied to other complex genomes to resolve the last gene-rich gaps, improve duplicate gene annotation, and better understand copy-number-variant genetic diversity at the base-pair level.

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April 21, 2020

FLAM-seq: full-length mRNA sequencing reveals principles of poly(A) tail length control.

Although messenger RNAs are key molecules for understanding life, until now, no method has existed to determine the full-length sequence of endogenous mRNAs including their poly(A) tails. Moreover, although non-A nucleotides can be incorporated in poly(A) tails, there also exists no method to accurately sequence them. Here, we present full-length poly(A) and mRNA sequencing (FLAM-seq), a rapid and simple method for high-quality sequencing of entire mRNAs. We report a complementary DNA library preparation method coupled to single-molecule sequencing to perform FLAM-seq. Using human cell lines, brain organoids and Caenorhabditis elegans we show that FLAM-seq delivers high-quality full-length mRNA sequences for thousands of different genes per sample. We find that 3′ untranslated region length is correlated with poly(A) tail length, that alternative polyadenylation sites and alternative promoters for the same gene are linked to different tail lengths, and that tails contain a substantial number of cytosines.

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April 21, 2020

Comprehensive evaluation of non-hybrid genome assembly tools for third-generation PacBio long-read sequence data.

Long reads obtained from third-generation sequencing platforms can help overcome the long-standing challenge of the de novo assembly of sequences for the genomic analysis of non-model eukaryotic organisms. Numerous long-read-aided de novo assemblies have been published recently, which exhibited superior quality of the assembled genomes in comparison with those achieved using earlier second-generation sequencing technologies. Evaluating assemblies is important in guiding the appropriate choice for specific research needs. In this study, we evaluated 10 long-read assemblers using a variety of metrics on Pacific Biosciences (PacBio) data sets from different taxonomic categories with considerable differences in genome size. The results allowed us to narrow down the list to a few assemblers that can be effectively applied to eukaryotic assembly projects. Moreover, we highlight how best to use limited genomic resources for effectively evaluating the genome assemblies of non-model organisms. © The Author 2017. Published by Oxford University Press.

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April 21, 2020

The genome of cultivated peanut provides insight into legume karyotypes, polyploid evolution and crop domestication.

High oil and protein content make tetraploid peanut a leading oil and food legume. Here we report a high-quality peanut genome sequence, comprising 2.54?Gb with 20 pseudomolecules and 83,709 protein-coding gene models. We characterize gene functional groups implicated in seed size evolution, seed oil content, disease resistance and symbiotic nitrogen fixation. The peanut B subgenome has more genes and general expression dominance, temporally associated with long-terminal-repeat expansion in the A subgenome that also raises questions about the A-genome progenitor. The polyploid genome provided insights into the evolution of Arachis hypogaea and other legume chromosomes. Resequencing of 52 accessions suggests that independent domestications formed peanut ecotypes. Whereas 0.42-0.47 million years ago (Ma) polyploidy constrained genetic variation, the peanut genome sequence aids mapping and candidate-gene discovery for traits such as seed size and color, foliar disease resistance and others, also providing a cornerstone for functional genomics and peanut improvement.

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April 21, 2020

Expedited assessment of terrestrial arthropod diversity by coupling Malaise traps with DNA barcoding 1.

Monitoring changes in terrestrial arthropod communities over space and time requires a dramatic increase in the speed and accuracy of processing samples that cannot be achieved with morphological approaches. The combination of DNA barcoding and Malaise traps allows expedited, comprehensive inventories of species abundance whose cost will rapidly decline as high-throughput sequencing technologies advance. Aside from detailing protocols from specimen sorting to data release, this paper describes their use in a survey of arthropod diversity in a national park that examined 21?194 specimens representing 2255 species. These protocols can support arthropod monitoring programs at regional, national, and continental scales.

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April 21, 2020

Transmission of ciprofloxacin resistance in Salmonella mediated by a novel type of conjugative helper plasmids.

Ciprofloxacin resistance in Salmonella has been increasingly reported due to the emergence and dissemination of multiple Plasmid-Mediated Quinolone Resistance (PMQR) determinants, which are mainly located in non-conjugative plasmids or chromosome. In this study, we aimed to depict the molecular mechanisms underlying the rare phenomenon of horizontal transfer of ciprofloxacin resistance phenotype in Salmonella by conjugation experiments, S1-PFGE and complete plasmid sequencing. Two types of non-conjugative plasmids, namely an IncX1 type carrying a qnrS1 gene, and an IncH1 plasmid carrying the oqxAB-qnrS gene, both ciprofloxacin resistance determinants in Salmonella, were recovered from two Salmonella strains. Importantly, these non-conjugative plasmids could be fused with a novel Incl1 type conjugative helper plasmid, which could target insertion sequence (IS) elements located in the non-conjugative, ciprofloxacin-resistance-encoding plasmid through replicative transcription, eventually forming a hybrid conjugative plasmid transmissible among members of Enterobacteriaceae. Since our data showed that such conjugative helper plasmids are commonly detectable among clinical Salmonella strains, particularly S. Typhimurium, fusion events leading to generation and enhanced dissemination of conjugative ciprofloxacin resistance-encoding plasmids in Salmonella are expected to result in a sharp increase in the incidence of resistance to fluoroquinolone, the key choice for treating life-threatening Salmonella infections, thereby posing a serious public health threat.

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