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Tuesday, April 21, 2020

An African Salmonella Typhimurium ST313 sublineage with extensive drug-resistance and signatures of host adaptation.

Bloodstream infections by Salmonella enterica serovar Typhimurium constitute a major health burden in sub-Saharan Africa (SSA). These invasive non-typhoidal (iNTS) infections are dominated by isolates of the antibiotic resistance-associated sequence type (ST) 313. Here, we report emergence of ST313 sublineage II.1 in the Democratic Republic of the Congo. Sublineage II.1 exhibits extensive drug resistance, involving a combination of multidrug resistance, extended spectrum ß-lactamase production and azithromycin resistance. ST313 lineage II.1 isolates harbour an IncHI2 plasmid we name pSTm-ST313-II.1, with one isolate also exhibiting decreased ciprofloxacin susceptibility. Whole genome sequencing reveals that ST313 II.1 isolates have accumulated genetic signatures potentially associated with…

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Tuesday, April 21, 2020

Complete Genome Sequence of Sequevar 14M Ralstonia solanacearum Strain HA4-1 Reveals Novel Type III Effectors Acquired Through Horizontal Gene Transfer.

Ralstonia solanacearum, which causes bacterial wilt in a broad range of plants, is considered a “species complex” due to its significant genetic diversity. Recently, we have isolated a new R. solanacearum strain HA4-1 from Hong’an county in Hubei province of China and identified it being phylotype I, sequevar 14M (phylotype I-14M). Interestingly, we found that it can cause various disease symptoms among different potato genotypes and display different pathogenic behavior compared to a phylogenetically related strain, GMI1000. To dissect the pathogenic mechanisms of HA4-1, we sequenced its whole genome by combined sequencing technologies including Illumina HiSeq2000, PacBio RS II, and…

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Tuesday, April 21, 2020

Long-Read Sequencing Emerging in Medical Genetics

The wide implementation of next-generation sequencing (NGS) technologies has revolutionized the field of medical genetics. However, the short read lengths of currently used sequencing approaches pose a limitation for identification of structural variants, sequencing repetitive regions, phasing alleles and distinguishing highly homologous genomic regions. These limitations may significantly contribute to the diagnostic gap in patients with genetic disorders who have undergone standard NGS, like whole exome or even genome sequencing. Now, the emerging long-read sequencing (LRS) technologies may offer improvements in the characterization of genetic variation and regions that are difficult to assess with the currently prevailing NGS approaches. LRS…

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Tuesday, April 21, 2020

Long-read based de novo assembly of low-complexity metagenome samples results in finished genomes and reveals insights into strain diversity and an active phage system.

Complete and contiguous genome assemblies greatly improve the quality of subsequent systems-wide functional profiling studies and the ability to gain novel biological insights. While a de novo genome assembly of an isolated bacterial strain is in most cases straightforward, more informative data about co-existing bacteria as well as synergistic and antagonistic effects can be obtained from a direct analysis of microbial communities. However, the complexity of metagenomic samples represents a major challenge. While third generation sequencing technologies have been suggested to enable finished metagenome-assembled genomes, to our knowledge, the complete genome assembly of all dominant strains in a microbiome sample…

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Wednesday, October 23, 2019

CRISPR/Cas9-generated p47(phox)-deficient cell line for Chronic Granulomatous Disease gene therapy vector development.

Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47(phox)-deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (?GT) mutation in p47(phox) encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47(phox)-subunit deficiency represents the second…

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Wednesday, October 23, 2019

A high quality assembly of the Nile Tilapia (Oreochromis niloticus) genome reveals the structure of two sex determination regions.

Tilapias are the second most farmed fishes in the world and a sustainable source of food. Like many other fish, tilapias are sexually dimorphic and sex is a commercially important trait in these fish. In this study, we developed a significantly improved assembly of the tilapia genome using the latest genome sequencing methods and show how it improves the characterization of two sex determination regions in two tilapia species.A homozygous clonal XX female Nile tilapia (Oreochromis niloticus) was sequenced to 44X coverage using Pacific Biosciences (PacBio) SMRT sequencing. Dozens of candidate de novo assemblies were generated and an optimal assembly…

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Sunday, September 22, 2019

Is there foul play in the leaf pocket? The metagenome of floating fern Azolla reveals endophytes that do not fix N2 but may denitrify.

Dinitrogen fixation by Nostoc azollae residing in specialized leaf pockets supports prolific growth of the floating fern Azolla filiculoides. To evaluate contributions by further microorganisms, the A. filiculoides microbiome and nitrogen metabolism in bacteria persistently associated with Azolla ferns were characterized. A metagenomic approach was taken complemented by detection of N2 O released and nitrogen isotope determinations of fern biomass. Ribosomal RNA genes in sequenced DNA of natural ferns, their enriched leaf pockets and water filtrate from the surrounding ditch established that bacteria of A. filiculoides differed entirely from surrounding water and revealed species of the order Rhizobiales. Analyses of seven cultivated…

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Sunday, September 22, 2019

Computational identification of novel genes: current and future perspectives.

While it has long been thought that all genomic novelties are derived from the existing material, many genes lacking homology to known genes were found in recent genome projects. Some of these novel genes were proposed to have evolved de novo, ie, out of noncoding sequences, whereas some have been shown to follow a duplication and divergence process. Their discovery called for an extension of the historical hypotheses about gene origination. Besides the theoretical breakthrough, increasing evidence accumulated that novel genes play important roles in evolutionary processes, including adaptation and speciation events. Different techniques are available to identify genes and…

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Sunday, September 22, 2019

wtf genes are prolific dual poison-antidote meiotic drivers.

Meiotic drivers are selfish genes that bias their transmission into gametes, defying Mendelian inheritance. Despite the significant impact of these genomic parasites on evolution and infertility, few meiotic drive loci have been identified or mechanistically characterized. Here, we demonstrate a complex landscape of meiotic drive genes on chromosome 3 of the fission yeasts Schizosaccharomyces kambucha and S. pombe. We identify S. kambucha wtf4 as one of these genes that acts to kill gametes (known as spores in yeast) that do not inherit the gene from heterozygotes. wtf4 utilizes dual, overlapping transcripts to encode both a gamete-killing poison and an antidote…

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Sunday, September 22, 2019

Cataloguing over-expressed genes in Epstein Barr Virus immortalized lymphoblastoid cell lines through consensus analysis of PacBio transcriptomes corroborates hypomethylation of chromosome 1

The ability of Epstein Barr Virus (EBV) to transform resting cell B-cells into immortalized lymphoblastoid cell lines (LCL) provides a continuous source of peripheral blood lymphocytes that are used to model conditions in which these lymphocytes play a key role. Here, the PacBio generated transcriptome of three LCLs from a parent-daughter trio (SRAid:SRP036136) provided by a previous study [1] were analyzed using a kmer-based version of YeATS (KEATS). The set of over-expressed genes in these cell lines were determined based on a comparison with the PacBio transcriptome of twenty tissues pro- vided by another study (hOPTRS) [2]. MIR155 long non-coding…

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Sunday, September 22, 2019

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics.

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio’s single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT…

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Sunday, September 22, 2019

Sixteen diverse laboratory mouse reference genomes define strain-specific haplotypes and novel functional loci.

We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene…

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Sunday, September 22, 2019

Extensive allele-specific translational regulation in hybrid mice.

Translational regulation is mediated through the interaction between diffusible trans-factors and cis-elements residing within mRNA transcripts. In contrast to extensively studied transcriptional regulation, cis-regulation on translation remains underexplored. Using deep sequencing-based transcriptome and polysome profiling, we globally profiled allele-specific translational efficiency for the first time in an F1 hybrid mouse. Out of 7,156 genes with reliable quantification of both alleles, we found 1,008 (14.1%) exhibiting significant allelic divergence in translational efficiency. Systematic analysis of sequence features of the genes with biased allelic translation revealed that local RNA secondary structure surrounding the start codon and proximal out-of-frame upstream AUGs could affect…

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Sunday, September 22, 2019

A single-molecule long-read survey of the human transcriptome.

Global RNA studies have become central to understanding biological processes, but methods such as microarrays and short-read sequencing are unable to describe an entire RNA molecule from 5′ to 3′ end. Here we use single-molecule long-read sequencing technology from Pacific Biosciences to sequence the polyadenylated RNA complement of a pooled set of 20 human organs and tissues without the need for fragmentation or amplification. We show that full-length RNA molecules of up to 1.5 kb can readily be monitored with little sequence loss at the 5′ ends. For longer RNA molecules more 5′ nucleotides are missing, but complete intron structures…

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Sunday, September 22, 2019

Comparative Annotation Toolkit (CAT)-simultaneous clade and personal genome annotation.

The recent introductions of low-cost, long-read, and read-cloud sequencing technologies coupled with intense efforts to develop efficient algorithms have made affordable, high-quality de novo sequence assembly a realistic proposition. The result is an explosion of new, ultracontiguous genome assemblies. To compare these genomes, we need robust methods for genome annotation. We describe the fully open source Comparative Annotation Toolkit (CAT), which provides a flexible way to simultaneously annotate entire clades and identify orthology relationships. We show that CAT can be used to improve annotations on the rat genome, annotate the great apes, annotate a diverse set of mammals, and annotate…

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