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August 1, 2019

Newly designed 16S rRNA metabarcoding primers amplify diverse and novel archaeal taxa from the environment.

High-throughput studies of microbial communities suggest that Archaea are a widespread component of microbial diversity in various ecosystems. However, proper quantification of archaeal diversity and community ecology remains limited, as sequence coverage of Archaea is usually low owing to the inability of available prokaryotic primers to efficiently amplify archaeal compared to bacterial rRNA genes. To improve identification and quantification of Archaea, we designed and validated the utility of several primer pairs to efficiently amplify archaeal 16S rRNA genes based on up-to-date reference genes. We demonstrate that several of these primer pairs amplify phylogenetically diverse Archaea with high sequencing coverage, outperforming…

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August 1, 2019

Complete Genome Sequence of Actinosynnema pretiosum X47, An Industrial Strain that Produces the Antibiotic Ansamitocin AP-3.

Ansamitocins are extraordinarily potent antitumor agents. Ansamitocin P-3 (AP-3), which is produced by Actinosynnema pretiosum, has been developed as a cytotoxic drug for breast cancer. Despite its importance, AP-3 is of limited applicability because of the low production yield. A. pretiosum strain X47 was developed from A. pretiosum ATCC 31565 by mutation breeding and shows a relatively high AP-3 yield. Here, we analyzed the A. pretiosum X47 genome, which is ~8.13 Mb in length with 6693 coding sequences, 58 tRNA genes, and 15 rRNA genes. The DNA sequence of the ansamitocin biosynthetic gene cluster is highly similar to that of the…

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August 1, 2019

Complete genome of Pseudoalteromonas atlantica ECSMB14104, a Gammaproteobacterium inducing mussel settlement

Pseudoalteromonas is widely distributed in the marine environments and the biofilms formed by Pseudoalteromonas promote settlement of many species of invertebrates. Here, we show the complete genome of Pseudoalteromonas atlantica ECSMB14104, which was isolated from biofilms formed in the East China Sea and exhibited inducing activity on the Mytilus coruscus settlement. Complete genome of this strain containsa total of 3325 genes and the GC content of 41.02%. This genomic information is contributed to molecular mechanism of P. atlantica ECSMB14104 regulating mussel settlement.

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August 1, 2019

Complete genome sequence of Bradymonas sediminis FA350T, the first representative of the order Bradymonadales

Bradymonas sediminis FA350T (=DSM 28820T?=?CICC 10904T) is a Gram-negative, rod-shaped and facultatively anaerobic bacterium isolated from coastal sediments from the Xiaoshi Island, Weihai, China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that that strain FA350T belonged to a novel bacterial order in the class Deltaproteobacteria. Then, based on polyphasic taxonomy analyses, a novel order Bradymonadales and a novel family Bradymonadaceae were proposed and validly published. Here, we reported the complete genome of this strain; the genome is 5,045,683?bp in size, has a GC content of 61.1% and contains 3992 predicted genes. Strain FA350T featured being able to prey…

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August 1, 2019

Genome Analysis of Carbaryl-Degrading Strain Pseudomonas putida XWY-1.

Carbaryl was a widely used pesticide in the agriculture industry. The toxicity against non-target organisms and the environmental pollution it caused became the focus of public concern. However, the microbial mechanism of carbaryl degradation was not fully investigated. In the study, we reported the complete genome of the carbaryl-degrading Pseudomonas putida strain XWY-1, which consists of a chromosome (5.9 Mbp) and a plasmid (0.4 Mbp). The carbaryl degradation genes are located on the plasmid. The study on the genome will facilitate to further elucidate the carbaryl degradation and advance the potential biotechnological applications of P. putida strain XWY-1.

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August 1, 2019

Comparative genome analysis provides novel insight into the interaction of Aquimarina sp. AD1, BL5 and AD10 with their macroalgal host.

The Aquimarina genus is widely distributed throughout the marine environment, however little is understood regarding its ecological role, particularly when in association with eukaryotic hosts. Here, we examine the genomes of two opportunistic pathogens, Aquimarina sp. AD1 and BL5, and a non-pathogenic strain Aquimarina sp. AD10, that were isolated from diseased individuals of the red alga Delisea pulchra. Each strain encodes multiple genes for the degradation of marine carbohydrates and vitamin biosynthesis. These traits are hypothesised to promote nutrient exchange between the Aquimarina strains and their algal host, facilitating a close symbiotic relationship. Moreover, each strain harbours the necessary genes…

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July 9, 2019

Single cell isoform sequencing (scIso-Seq) identifies novel full-length mRNAs and cell type-specific expression

Single cell RNA-seq (scRNA-seq) is an emerging field for characterizing cell heterogeneity in complex tissues. However, most scRNA-seq methodologies are limited to gene count information due to short read lengths. Here, we combine the microfluidics scRNA-seq technique, Drop-Seq, with PacBio Single Molecule, Real-Time (SMRT) Sequencing to generate full-length transcript isoforms that can be confidently assigned to individual cells. We generated single cell Iso-Seq (scIso-Seq) libraries for chimp and human cerebral organoid samples on the Dolomite Nadia platform and sequenced each library with two SMRT Cells 8M on the PacBio Sequel II System. We developed a bioinformatics pipeline to identify, classify,…

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July 1, 2019

High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution.

Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members…

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July 1, 2019

Agricultural intensification reduces microbial network complexity and the abundance of keystone taxa in roots.

Root-associated microbes play a key role in plant performance and productivity, making them important players in agroecosystems. So far, very few studies have assessed the impact of different farming systems on the root microbiota and it is still unclear whether agricultural intensification influences the structure and complexity of microbial communities. We investigated the impact of conventional, no-till, and organic farming on wheat root fungal communities using PacBio SMRT sequencing on samples collected from 60 farmlands in Switzerland. Organic farming harbored a much more complex fungal network with significantly higher connectivity than conventional and no-till farming systems. The abundance of keystone…

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July 1, 2019

Single-Cell Virus Sequencing of Influenza Infections That Trigger Innate Immunity.

Influenza virus-infected cells vary widely in their expression of viral genes and only occasionally activate innate immunity. Here, we develop a new method to assess how the genetic variation in viral populations contributes to this heterogeneity. We do this by determining the transcriptome and full-length sequences of all viral genes in single cells infected with a nominally "pure" stock of influenza virus. Most cells are infected by virions with defects, some of which increase the frequency of innate-immune activation. These immunostimulatory defects are diverse and include mutations that perturb the function of the viral polymerase protein PB1, large internal deletions…

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July 1, 2019

Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the 16S-ITS-23S rRNA operon.

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The…

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July 1, 2019

Dissemination of multiple carbapenem resistance genes in an in vitro gut model simulating the human colon.

Carbapenemase-producing Enterobacteriaceae (CPE) pose a major global health risk. Mobile genetic elements account for much of the increasing CPE burden.To investigate CPE colonization and the impact of antibiotic exposure on subsequent resistance gene dissemination within the gut microbiota using a model to simulate the human colon.Gut models seeded with CPE-negative human faeces [screened with BioMérieux chromID® CARBA-SMART (Carba-Smart), Cepheid Xpert® Carba-R assay (XCR)] were inoculated with distinct carbapenemase-producing Klebsiella pneumoniae strains (KPC, NDM) and challenged with imipenem or piperacillin/tazobactam then meropenem. Resistant populations were enumerated daily on selective agars (Carba-Smart); CPE genes were confirmed by PCR (XCR, Check-Direct CPE Screen…

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July 1, 2019

Different knockout genotypes of OsIAA23 in rice using CRISPR/Cas9 generating different phenotypes.

We have isolated several Osiaa23 rice mutants with different knockout genotypes, resulting in different phenotypes, which suggested that different genetic backgrounds or mutation types influence gene function. The Auxin/Indole-3-Acetic Acid (Aux/IAA) gene family performs critical roles in auxin signal transduction in plants. In rice, the gene OsIAA23 (Os06t0597000) is known to affect development of roots and shoots, but previous knockouts in OsIAA23 have been sterile and difficult for research continuously. Here, we isolate new Osiaa23 mutants using the CRISPR/Cas9 system in japonica (Wuyunjing24) and indica (Kasalath) rice, with extensive genome re-sequencing to confirm the absence of off-target effects. In Kasalath,…

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