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August 12, 2019

Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome

The DNA sequencing technologies in use today produce either highly accurate short reads or less-accurate long reads. We report the optimization of circular consensus sequencing (CCS) to improve the accuracy of single-molecule real-time (SMRT) sequencing (PacBio) and generate highly accurate (99.8%) long high-fidelity (HiFi) reads with an average length of 13.5 kilobases (kb). We applied our approach to sequence the well-characterized human HG002/NA24385 genome and obtained precision and recall rates of at least 99.91% for single-nucleotide variants (SNVs), 95.98% for insertions and deletions <50 bp (indels) and 95.99% for structural variants. Our CCS method matches or exceeds the ability of short-read…

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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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July 19, 2019

Application Brief: Targeted sequencing for amplicons – Best Practices

With Single Molecule, Real-Time (SMRT) Sequencing and the Sequel System, you can easily and cost effectively generate highly accurate long reads (HiFi reads, >99% single-molecule accuracy) from genes or regions of interest ranging in size from several hundred base pairs to 20 kb. Target all types of variation across relevant genomic regions, including low complexity regions like repeat expansions, promoters, and flanking regions of transposable elements.

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July 17, 2019

Webinar: Variant calling and de novo genome assembly with PacBio HiFi reads

In this webinar, Sarah Kingan, Staff Scientist, PacBio, presents recent work on de novo genome assembly using PacBio HiFi reads. She highlights the benefits of HiFi data for base level accuracy, haplotype phasing, and ease of computation. And in samples ranging from human to plants, she benchmarks various tools for HiFi assembly and phasing, including the newly extended FALCON-Unzip assembler. Subsequently, Andrew Carroll, Genomics Product Lead, GoogleAI, explores how the GoogleAI team retrained DeepVariant, a highly accurate SNP and Indel caller, for PacBio HiFi data. The resulting DeepVariant models achieve comparable accuracies to short-read methods with the additional benefit of…

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July 1, 2019

Identification and characterisation of anti – Pseudomonas aeruginosa proteins in mucus of the brown garden snail, Cornu aspersum.

Background: Novel antimicrobial treatments are urgently needed. Previous work has shown that the mucus of the brown garden snail (Cornu aspersum) has antimicrobial properties, in particular against type culture collection strains of Pseudomonas aeruginosa. We hypothesised that it would also be effective against clinical isolates of the bacterium and that investigation of fractions of the mucus would identify one or more proteins with anti-pseudomonal properties, which could be further characterised. Materials and methods: Mucus was extracted from snails collected from the wild. Antimicrobial activity against laboratory and clinical isolates of Ps. aeruginosa was determined in disc diffusion assays. Mucus was…

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July 1, 2019

Genome sequence resource for Ilyonectria mors-panacis, causing rusty root rot of Panax notoginseng.

Ilyonectria mors-panacis is a serious disease hampering the production of Panax notoginseng, an important Chinese medicinal herb, widely used for its anti-inflammatory, anti-fatigue, hepato-protective, and coronary heart disease prevention effects. Here, we report the first Illumina-Pacbio hybrid sequenced draft genome assembly of I. mors-panacis strain G3B and its annotation. The availability of this genome sequence not only represents an important tool toward understanding the genetics behind the infection mechanism of I. mors-panacis strain G3B but also will help illuminate the complexities of the taxonomy of this species.

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July 1, 2019

Chromosomal-level assembly of the blolsod clam, Scapharca (Anadara) broughtonii, using long sequence reads and Hi-C.

The blood clam, Scapharca (Anadara) broughtonii, is an economically and ecologically important marine bivalve of the family Arcidae. Efforts to study their population genetics, breeding, cultivation, and stock enrichment have been somewhat hindered by the lack of a reference genome. Herein, we report the complete genome sequence of S. broughtonii, a first reference genome of the family Arcidae.A total of 75.79 Gb clean data were generated with the Pacific Biosciences and Oxford Nanopore platforms, which represented approximately 86× coverage of the S. broughtonii genome. De novo assembly of these long reads resulted in an 884.5-Mb genome, with a contig N50…

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July 1, 2019

Chromosome-level genome assembly of Triplophysa tibetana, a fish adapted to the harsh high-altitude environment of the Tibetan Plateau.

Triplophysa is an endemic fish genus of the Tibetan Plateau in China. Triplophysa tibetana, which lives at a recorded altitude of ~4,000 m and plays an important role in the highland aquatic ecosystem, serves as an excellent model for investigating high-altitude environmental adaptation. However, evolutionary and conservation studies of T. tibetana have been limited by scarce genomic resources for the genus Triplophysa. In the present study, we applied PacBio sequencing and the Hi-C technique to assemble the T. tibetana genome. A 652-Mb genome with 1,325 contigs with an N50 length of 3.1 Mb was obtained. The 1,137 contigs were further assembled into 25 chromosomes,…

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July 1, 2019

Varieties of immunity activities and gut contents in tilapia with seasonal changes.

We performed 16S rDNA sequencing of tilapia fecal samples to analyze changes in tilapia gut contents after cultivation of the fish in the presence of sandwich-like floating beds of Chinese medicinal herbs (5 and 10% planting-areas; 5% Polygonum cuspidatum). The interactive effects between water quality and blood and hepatic pro- and anti-inflammatory concentrations were also assessed. Our results showed that the water quality (i.e., NO3--N, NO2--N, TP removal rates) improved, and the abundance of Chloroflexi and Cyanobacteria increased. The abundance of Bacteroidetes, Verrucomicrobia, Saccharibacteria, and Actinobacteria showed both significant seasonal decreases and increases in the presence of P. cuspidatum (increases…

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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

Full-length 16S rRNA gene classification of Atlantic salmon bacteria and effects of using different 16S variable regions on community structure analysis.

Understanding fish-microbial relationships may be of great value for fish producers as fish growth, development and welfare are influenced by the microbial community associated with the rearing systems and fish surfaces. Accurate methods to generate and analyze these microbial communities would be an important tool to help improve understanding of microbial effects in the industry. In this study, we performed taxonomic classification and determination of operational taxonomic units on Atlantic salmon microbiota by taking advantage of full-length 16S rRNA gene sequences. Skin mucus was dominated by the genera Flavobacterium and Psychrobacter. Intestinal samples were dominated by the genera Carnobacterium, Aeromonas,…

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July 1, 2019

Snf2 controls pulcherriminic acid biosynthesis and antifungal activity of the biocontrol yeast Metschnikowia pulcherrima.

Metschnikowia pulcherrima synthesises the pigment pulcherrimin, from cyclodileucine (cyclo(Leu-Leu)) as a precursor, and exhibits strong antifungal activity against notorious plant pathogenic fungi. This yeast therefore has great potential for biocontrol applications against fungal diseases; particularly in the phyllosphere where this species is frequently found. To elucidate the molecular basis of the antifungal activity of M. pulcherrima, we compared a wild-type strain with a spontaneously occurring, pigmentless, weakly antagonistic mutant derivative. Whole genome sequencing of the wild-type and mutant strains identified a point mutation that creates a premature stop codon in the transcriptional regulator gene SNF2 in the mutant. Complementation of…

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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

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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