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Tuesday, June 1, 2021

New discoveries from closing Salmonella genomes using Pacific Biosciences continuous long reads.

The newer hierarchical genome assembly process (HGAP) performs de novo assembly using data from a single PacBio long insert library. To assess the benefits of this method, DNA from several Salmonella enterica serovars was isolated from a pure culture. Genome sequencing was performed using Pacific Biosciences RS sequencing technology. The HGAP process enabled us to close sixteen Salmonella subsp. enterica genomes and their associated mobile elements: The ten serotypes include: Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) S. Bareilly, S. Heidelberg, S. Cubana, S. Javiana and S. Typhimurium, S. Newport, S. Montevideo, S. Agona, and S. Tennessee. In addition,…

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Tuesday, June 1, 2021

Genome analysis of a bacterium that causes lameness.

Lameness is a significant problem resulting in millions of dollars in lost revenue annually. In commercial broilers, the most common cause of lameness is bacterial chondronecrosis with osteomyelitis (BCO). We are using a wire flooring model to induce lameness attributable to BCO. We used 16S ribosomal DNA sequencing to determine that Staphylococcus spp. were the main species associated with BCO. Staphylococcus agnetis, which previously had not been isolated from poultry, was the principal species isolated from the majority of the bone lesion samples. Administering S. agnetis in the drinking water to broilers reared on wire flooring increased the incidence of…

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Tuesday, June 1, 2021

SMRT Sequencing of the alala genome

Single Molecule Real-Time (SMRT) Sequencing was used to generate long reads for whole genome shotgun sequencing of the genome of the`alala (Hawaiian crow). The ‘alala is endemic to Hawaii, and the only surviving lineage of the crow family, Corvidae, in the Hawaiian Islands. The population declined to less than 20 individuals in the 1990s, and today this charismatic species is extinct in the wild. Currently existing in only two captive breeding facilities, reintroduction of the ‘alala is scheduled to begin in the Fall of 2016. Reintroduction efforts will be assisted by information from the ‘alala genome generated and assembled by…

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Tuesday, June 1, 2021

High-quality de novo genome assembly and intra-individual mitochondrial instability in the critically endangered kakapo

The kakapo (Strigops habroptila) is a large, flightless parrot endemic to New Zealand. It is highly endangered with only ~150 individuals remaining, and intensive conservation efforts are underway to save this iconic species from extinction. These include genetic studies to understand critical genes relevant to fertility, adaptation and disease resistance, and genetic diversity across the remaining population for future breeding program decisions. To aid with these efforts, we have generated a high-quality de novo genome assembly using PacBio long-read sequencing. Using the new diploid-aware FALCON-Unzip assembler, the resulting genome of 1.06 Gb has a contig N50 of 5.6 Mb (largest…

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Friday, February 5, 2021

Webinar: SMRT Sequencing applications in plant and animal sciences: an overview

In this webinar, Emily Hatas of PacBio shares information about the applications and benefits of SMRT Sequencing in plant and animal biology, agriculture, and industrial research fields. This session contains an overview of several applications: whole-genome sequencing for de novo assembly; transcript isoform sequencing (Iso-Seq) method for genome annotation; targeted sequencing solutions; and metagenomics and microbial interactions. High-level workflows and best practices are discussed for key applications.

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Friday, February 5, 2021

Webinar: Using Iso-Seq analysis to build a better annotation

Long-read sequencing technologies like Iso-Seq analysis present researchers with a powerful tool for probing the transcriptomes of many species. The ability to sequence transcripts from end-to-end has revealed transcription complexity on a scale that was previously impossible. This sequence rich information has also improved our ability to predict transcript functions and biotypes. Researchers can now use Iso-Seq analysis to discover transcript models in almost any species with an accuracy on par with human and mouse annotations. In this webinar, Richard Kuo discusses the core concepts behind Iso-Seq analysis and how to use it to improve or build a new transcriptome…

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Friday, February 5, 2021

ASHG PacBio Workshop: The Iso-Seq method for discovering alternative splicing in human diseases

In this ASHG workshop presentation, Elizabeth Tseng of PacBio showed how the Iso-Seq method can be used to discover disease-associated alternative splicing. Because this approach to isoform sequencing yields accurate, full-length transcripts requiring no assembly, it’s ideal for disease studies that need a more comprehensive picture of alternative splicing activity. Tseng offered several published examples of how the Iso-Seq method has been used for everything from single-gene studies to whole-transcriptome studies, and also detailed how the latest Sequel System chemistry recovers more genes and produces more usable reads.

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Friday, February 5, 2021

PAG Conference: Iso-Seq analysis for plant & animal genomes – annotation evaluation & phasing

In this presentation, Elizabeth Tseng explains how PacBio’s full-length RNA Sequencing using the Iso-Seq method can characterize full-length transcripts without the need for computational transcript assembly. The Iso-Seq method is fully supported bioinformatically through PacBio’s SMRT Analysis software that outputs high-quality, full-length transcript sequences that can be used for genome annotation and novel gene discovery. Elizabeth shows that the highly accurate reads can be used to discover allelic-specific isoform expressions in transcriptome data.

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Friday, February 5, 2021

User Group Meeting: No Assembly Required – Making the most of Iso-Seq data

In this PacBio User Group Meeting presentation, PacBio scientist Kristin Mars speaks about recent updates, such as the single-day library prep that’s now possible with the Iso-Seq Express workflow. She also notes that one SMRT Cell 8M is sufficient for most Iso-Seq experiments for whole transcriptome sequencing at an affordable price.

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Tuesday, April 21, 2020

Identification and characterization of chicken circovirus from commercial broiler chickens in China.

Circoviruses are found in many species, including mammals, birds, lower vertebrates and invertebrates. To date, there are no reports of circovirus-induced diseases in chickens. In this study, we identified a new strain of chicken circovirus (CCV) by PacBio third-generation sequencing samples from chickens with acute gastroenteritis in a Shandong commercial broiler farm in China. The complete genome of CCV was verified by inverse PCR. Genomic analysis revealed that CCV codes two inverse open reading frames (ORFs), and a potential stem-loop structure was present at the 5′ end with a structure typical of a circular virus. Phylogenetic tree analysis showed that…

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Tuesday, April 21, 2020

Chromosome-length haplotigs for yak and cattle from trio binning assembly of an F1 hybrid

Background Assemblies of diploid genomes are generally unphased, pseudo-haploid representations that do not correctly reconstruct the two parental haplotypes present in the individual sequenced. Instead, the assembly alternates between parental haplotypes and may contain duplications in regions where the parental haplotypes are sufficiently different. Trio binning is an approach to genome assembly that uses short reads from both parents to classify long reads from the offspring according to maternal or paternal haplotype origin, and is thus helped rather than impeded by heterozygosity. Using this approach, it is possible to derive two assemblies from an individual, accurately representing both parental contributions…

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Tuesday, April 21, 2020

Plasmid-encoded tet(X) genes that confer high-level tigecycline resistance in Escherichia coli.

Tigecycline is one of the last-resort antibiotics to treat complicated infections caused by both multidrug-resistant Gram-negative and Gram-positive bacteria1. Tigecycline resistance has sporadically occurred in recent years, primarily due to chromosome-encoding mechanisms, such as overexpression of efflux pumps and ribosome protection2,3. Here, we report the emergence of the plasmid-mediated mobile tigecycline resistance mechanism Tet(X4) in Escherichia coli isolates from China, which is capable of degrading all tetracyclines, including tigecycline and the US FDA newly approved eravacycline. The tet(X4)-harbouring IncQ1 plasmid is highly transferable, and can be successfully mobilized and stabilized in recipient clinical and laboratory strains of Enterobacteriaceae bacteria. It…

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Tuesday, April 21, 2020

RNA sequencing: the teenage years.

Over the past decade, RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing of mRNAs. However, as next-generation sequencing technologies have developed, so too has RNA-seq. Now, RNA-seq methods are available for studying many different aspects of RNA biology, including single-cell gene expression, translation (the translatome) and RNA structure (the structurome). Exciting new applications are being explored, such as spatial transcriptomics (spatialomics). Together with new long-read and direct RNA-seq technologies and better computational tools for data analysis, innovations in RNA-seq are contributing to a fuller understanding of RNA biology, from questions…

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