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Friday, February 26, 2021

A genome assembly of the domestic goat from 70x coverage of single molecule, real-time sequence.

Goat is an important source of milk, meat, and fiber, especially in developing countries. An advantage of goats as livestock is the low maintenance requirements and high adaptability compared to other milk producers. The global population of domestic goats exceeds 800 million. In Africa, goat production is characterized by low productivity levels, and attempts to introduce more productive breeds have met with poor success due in part to nutritional constraints. It has been suggested that incorporation of selective breeding within the herds adapted for survival could represent one approach to improving food security across Africa. A recently produced genome assembly…

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Friday, February 26, 2021

High-quality, highly contiguous re-assembly of the pig genome

Many applications of high throughput sequencing rely on the availability of an accurate reference genome. Errors in the reference genome assembly increase the number of false-positives in downstream analyses. Recently, we have shown that over 33% of the current pig reference genome, Sscrofa10.2, is either misassembled or otherwise unreliable for genomic analyses. Additionally, ~10% of the bases in the assembly are Ns in gaps of an arbitrary size. Thousands of highly fragmented contigs remain unplaced and many genes are known to be missing from the assembly. Here we present a new assembly of the pig genome, Sscrofa11, assembled using 65X…

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Friday, February 26, 2021

A complete solution for high-quality genome annotation using the PacBio Iso-Seq method

The PacBio Iso-Seq method produces high-quality, full-length transcripts of up to 10 kb and longer and has been used to annotate many important plant and animal genomes. We describe here the full Iso-Seq ecosystem that enables researchers to achieve high-quality genome annotations. The Iso-Seq Express workflow is a 1-day protocol that requires only 60-300 ng of total RNA and supports multiplexing of different tissues. Sequencing on a single SMRT Cell 8M on the Sequel II System produces up to 4 million full-length reads, sufficient to exhaustively characterize a whole transcriptome on the order of 15,000-17,000 genes with 100,000 or more…

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Saturday, February 20, 2021

Application Brief: Long-read RNA sequencing – Best Practices

With Single Molecule, Real-Time (SMRT) Sequencing and the Sequel Systems, you can easily and affordably sequence complete transcript isoforms in genes of interest or across the entire transcriptome. The Iso-Seq method allows users to generate full-length cDNA sequences up to 10 kb in length — with no assembly required — to confidently characterize full-length transcript isoforms.

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Saturday, February 20, 2021

Informational Guide: What’s the value of sequencing full-length RNA transcripts?

The study of genomics has revolutionized our understanding of science, but the field of transcriptomics grew with the need to explore the functional impacts of genetic variation. While different tissues in an organism may share the same genomic DNA, they can differ greatly in what regions are transcribed into RNA and in their patterns of RNA processing. By reviewing the history of transcriptomics, we can see the advantages of RNA sequencing using a full-length transcript approach become clearer.

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Friday, February 5, 2021

i5K Webinar: High-quality de novo insect genome assemblies using PacBio sequencing

PacBio Sequencing is characterized by very long sequence reads (averaging > 10,000 bases), lack of GC-bias, and high consensus accuracy. These features have allowed the method to provide a new gold standard in de novo genome assemblies, producing highly contiguous (contig N50 > 1 Mb) and accurate (> QV 50) genome assemblies. We will briefly describe the technology and then highlight the full workflow, from sample preparation through sequencing to data analysis, on examples of insect genome assemblies, and illustrate the difference these high-quality genomes represent with regard to biological insights, compared to fragmented draft assemblies generated by short-read sequencing.

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Friday, February 5, 2021

Educational Video: Step 1 – from sample to sequencing data for de novo assembly

This video provides an overview of the techniques and steps of preparing samples, DNA, and libraries for PacBio Single Molecule, Real-Time (SMRT) Sequencing to be used in de novo assembly projects. In this video, a PacBio scientist covers how to assess DNA quantity and purity, size-selection of DNA libraries, and provides and introduction to SMRT Sequencing, including the benefits of long-reads when generating high-quality genome assemblies.

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Friday, February 5, 2021

Webinar: Survey of transcriptome diversity using Iso-Seq analysis

The Iso-Seq method enables the sequencing of transcript isoforms from the 5’ end to their poly-A tails, eliminating the need for transcript reconstruction and inference. This webinar provides a comprehensive guide to Iso-Seq method data analysis, bioinformatics, and review key applications.

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Tuesday, April 21, 2020

An improved pig reference genome sequence to enable pig genetics and genomics research

The domestic pig (Sus scrofa) is important both as a food source and as a biomedical model with high anatomical and immunological similarity to humans. The draft reference genome (Sscrofa10.2) represented a purebred female pig from a commercial pork production breed (Duroc), and was established using older clone-based sequencing methods. The Sscrofa10.2 assembly was incomplete and unresolved redundancies, short range order and orientation errors and associated misassembled genes limited its utility. We present two highly contiguous chromosome-level genome assemblies created with more recent long read technologies and a whole genome shotgun strategy, one for the same Duroc female (Sscrofa11.1) and…

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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

Complete genome sequence of Bacillus velezensis JT3-1, a microbial germicide isolated from yak feces

Bacillus velezensis JT3-1 is a probiotic strain isolated from feces of the domestic yak (Bos grunniens) in the Gansu province of China. It has strong antagonistic activity against Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella Typhimurium, Mannheimia haemolytica, Staphylococcus hominis, Clostridium perfringens, and Mycoplasma bovis. These properties have made the JT3-1 strain the focus of commercial interest. In this study, we describe the complete genome sequence of JT3-1, with a genome size of 3,929,799 bp, 3761 encoded genes and an average GC content of 46.50%. Whole genome sequencing of Bacillus velezensis JT3-1 will lay a good foundation for elucidation of…

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Tuesday, April 21, 2020

Large-scale ruminant genome sequencing provides insights into their evolution and distinct traits.

The ruminants are one of the most successful mammalian lineages, exhibiting morphological and habitat diversity and containing several key livestock species. To better understand their evolution, we generated and analyzed de novo assembled genomes of 44 ruminant species, representing all six Ruminantia families. We used these genomes to create a time-calibrated phylogeny to resolve topological controversies, overcoming the challenges of incomplete lineage sorting. Population dynamic analyses show that population declines commenced between 100,000 and 50,000 years ago, which is concomitant with expansion in human populations. We also reveal genes and regulatory elements that possibly contribute to the evolution of the…

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Tuesday, April 21, 2020

Increased prevalence of Escherichia coli strains from food carrying blaNDM and mcr-1-bearing plasmids that structurally resemble those of clinical strains, China, 2015 to 2017.

Introduction: Emergence of resistance determinants of blaNDM and mcr-1 has undermined the antimicrobial effectiveness of the last line drugs carbapenems and colistin. Aim: This work aimed to assess the prevalence of blaNDM and mcr-1 in E. coli strains collected from food in Shenzhen, China, during the period 2015 to 2017. Methods: Multidrug-resistant E. coli strains were isolated from food samples. Plasmids encoding mcr-1 or blaNDM genes were characterised and compared with plasmids found in clinical isolates.ResultsAmong 1,166 non-repeated cephalosporin-resistant E. coli strains isolated from 2,147 food samples, 390 and 42, respectively, were resistant to colistin and meropenem, with five strains…

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Tuesday, April 21, 2020

Conjugal Transfer, Whole-Genome Sequencing, and Plasmid Analysis of Four mcr-1-Bearing Isolates from U.S. Patients.

Four Enterobacteriaceae clinical isolates bearing mcr-1 gene-harboring plasmids were characterized. All isolates demonstrated the ability to transfer colistin resistance to Escherichia coli; plasmids were stable in conjugants after multiple passages on nonselective media. mcr-1 was located on an IncX4 (n?=?3) or IncN (n?=?1) plasmid. The IncN plasmid harbored 13 additional antimicrobial resistance genes. Results indicate that the mcr-1-bearing plasmids in this study were highly transferable in vitro and stable in the recipients.This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

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Tuesday, April 21, 2020

Detection of VIM-1-Producing Enterobacter cloacae and Salmonella enterica Serovars Infantis and Goldcoast at a Breeding Pig Farm in Germany in 2017 and Their Molecular Relationship to Former VIM-1-Producing S. Infantis Isolates in German Livestock Production.

In 2011, VIM-1-producing Salmonella enterica serovar Infantis and Escherichia coli were isolated for the first time in four German livestock farms. In 2015/2016, highly related isolates were identified in German pig production. This raised the issue of potential reservoirs for these isolates, the relation of their mobile genetic elements, and potential links between the different affected farms/facilities. In a piglet-producing farm suspicious for being linked to some blaVIM-1 findings in Germany, fecal and environmental samples were examined for the presence of carbapenemase-producing Enterobacteriaceae and Salmonella spp. Newly discovered isolates were subjected to Illumina whole-genome sequencing (WGS) and S1 pulsed-field gel…

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