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July 7, 2019  |  

Resistance to ceftazidime-avibactam is due to tranposition of KPC in a porin-deficient strain of Klebsiella pneumoniae with increased efflux activity.

Ceftazidime-avibactam is an antibiotic with activity against serine beta-lactamases, including Klebsiella pneumoniae carbapenemase (KPC). Recently, reports have emerged of KPC-producing isolates resistant to this antibiotic, including a report of a wild-type KPC-3 producing sequence type 258 Klebsiella pneumoniae that was resistant to ceftazidime-avibactam. We describe a detailed analysis of this isolate, in the context of two other closely related KPC-3 producing isolates, recovered from the same patient. Both isolates encoded a nonfunctional OmpK35, whereas we demonstrate that a novel T333N mutation in OmpK36, present in the ceftazidime-avibactam resistant isolate, reduced the activity of this porin and impacted ceftazidime-avibactam susceptibility. In addition, we demonstrate that the increased expression of blaKPC-3 and blaSHV-12 observed in the ceftazidime-avibactam-resistant isolate was due to transposition of the Tn4401 transposon harboring blaKPC-3 into a second plasmid, pIncX3, which also harbored blaSHV-12, ultimately resulting in a higher copy number of blaKPC-3 in the resistant isolate. pIncX3 plasmid from the ceftazidime-avibactam resistant isolate, conjugated into a OmpK35/36-deficient K. pneumoniae background that harbored a mutation to the ramR regulator of the acrAB efflux operon recreated the ceftazidime-avibactam-resistant MIC of 32 µg/ml, confirming that this constellation of mutations is responsible for the resistance phenotype. Copyright © 2017 American Society for Microbiology.

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July 7, 2019  |  

Length-independent DNA packing into nanopore zero-mode waveguides for low-input DNA sequencing.

Compared with conventional methods, single-molecule real-time (SMRT) DNA sequencing exhibits longer read lengths than conventional methods, less GC bias, and the ability to read DNA base modifications. However, reading DNA sequence from sub-nanogram quantities is impractical owing to inefficient delivery of DNA molecules into the confines of zero-mode waveguides-zeptolitre optical cavities in which DNA sequencing proceeds. Here, we show that the efficiency of voltage-induced DNA loading into waveguides equipped with nanopores at their floors is five orders of magnitude greater than existing methods. In addition, we find that DNA loading is nearly length-independent, unlike diffusive loading, which is biased towards shorter fragments. We demonstrate here loading and proof-of-principle four-colour sequence readout of a polymerase-bound 20,000-base-pair-long DNA template within seconds from a sub-nanogram input quantity, a step towards low-input DNA sequencing and mammalian epigenomic mapping of native DNA samples.

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July 7, 2019  |  

Comparative genomic analysis of two clonally related multidrug resistant Mycobacterium tuberculosis by Single Molecule Real Time Sequencing.

Background: Multidrug-resistant tuberculosis (MDR-TB) is posing a major threat to global TB control. In this study, we focused on two consecutive MDR-TB isolated from the same patient before and after the initiation of anti-TB treatment. To better understand the genomic characteristics of MDR-TB, Single Molecule Real-Time (SMRT) Sequencing and comparative genomic analyses was performed to identify mutations that contributed to the stepwise development of drug resistance and growth fitness in MDR-TB underin vivochallenge of anti-TB drugs.Result:Both pre-treatment and post-treatment strain demonstrated concordant phenotypic and genotypic susceptibility profiles toward rifampicin, pyrazinamide, streptomycin, fluoroquinolones, aminoglycosides, cycloserine, ethionamide, and para-aminosalicylic acid. However, although both strains carried identical missense mutations atrpoBS531L,inhAC-15T, andembBM306V, MYCOTB Sensititre assay showed that the post-treatment strain had 16-, 8-, and 4-fold elevation in the minimum inhibitory concentrations (MICs) toward rifabutin, isoniazid, and ethambutol respectively. The results have indicated the presence of additional resistant-related mutations governing the stepwise development of MDR-TB. Further comparative genomic analyses have identified three additional polymorphisms between the clinical isolates. These include a single nucleotide deletion at nucleotide position 360 ofrv0888in pre-treatment strain, and a missense mutation atrv3303c(lpdA)V44I and a 6-bp inframe deletion at codon 67-68 inrv2071c(cobM)in the post-treatment strain. Multiple sequence alignment showed that these mutations were occurring at highly conserved regions among pathogenic mycobacteria. Using structural-based and sequence-based algorithms, we further predicted that the mutations potentially have deleterious effect on protein function.Conclusion:This is the first study that compared the full genomes of two clonally-related MDR-TB clinical isolates during the course of anti-TB treatment. Our work has demonstrated the robustness of SMRT Sequencing in identifying mutations among MDR-TB clinical isolates. Comparative genome analysis also suggested novel mutations atrv0888, lpdA, andcobMthat might explain the difference in antibiotic resistance and growth pattern between the two MDR-TB strains.

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July 7, 2019  |  

Analysis of hepatitis C NS5A resistance associated polymorphisms using ultra deep single molecule real time (SMRT) sequencing.

Development of Hepatitis C virus (HCV) resistance against direct-acting antivirals (DAAs), including NS5A inhibitors, is an obstacle to successful treatment of HCV when DAAs are used in sub-optimal combinations. Furthermore, it has been shown that baseline (pre-existing) resistance against DAAs is present in treatment naïve-patients and this will potentially complicate future treatment strategies in different HCV genotypes (GTs). Thus the aim was to detect low levels of NS5A resistant associated variants (RAVs) in a limited sample set of treatment-naïve patients of HCV GT1a and 3a, since such polymorphisms can display in vitro resistance as high as 60000 fold. Ultra-deep single molecule real time (SMRT) sequencing with the Pacific Biosciences (PacBio) RSII instrument was used to detect these RAVs. The SMRT sequencing was conducted on ten samples; three of them positive with Sanger sequencing (GT1a Q30H and Y93N, and GT3a Y93H), five GT1a samples, and two GT3a non-positive samples. The same methods were applied to the HCV GT1a H77-plasmid in a dilution series, in order to determine the error rates of replication, which in turn was used to determine the limit of detection (LOD), as defined by mean + 3SD, of minority variants down to 0.24%. We found important baseline NS5A RAVs at levels between 0.24 and 0.5%, which could potentially have clinical relevance. This new method with low level detection of baseline RAVs could be useful in predicting the most cost-efficient combination of DAA treatment, and reduce the treatment duration for an HCV infected individual. Copyright © 2015 Elsevier B.V. All rights reserved.

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July 7, 2019  |  

Resolving complex structural genomic rearrangements using a randomized approach.

Complex chromosomal rearrangements are structural genomic alterations involving multiple instances of deletions, duplications, inversions, or translocations that co-occur either on the same chromosome or represent different overlapping events on homologous chromosomes. We present SVelter, an algorithm that identifies regions of the genome suspected to harbor a complex event and then resolves the structure by iteratively rearranging the local genome structure, in a randomized fashion, with each structure scored against characteristics of the observed sequencing data. SVelter is able to accurately reconstruct complex chromosomal rearrangements when compared to well-characterized genomes that have been deeply sequenced with both short and long reads.

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July 7, 2019  |  

Finished genome sequence and methylome of the cyanide-degrading Pseudomonas pseudoalcaligenes strain CECT5344 as resolved by single-molecule real-time sequencing.

Pseudomonas pseudoalcaligenes CECT5344 tolerates cyanide and is also able to utilize cyanide and cyano-derivatives as a nitrogen source under alkaline conditions. The strain is considered as candidate for bioremediation of habitats contaminated with cyanide-containing liquid wastes. Information on the genome sequence of the strain CECT5344 became available previously. The P. pseudoalcaligenes CECT5344 genome was now resequenced by applying the single molecule, real-time (SMRT(®)) sequencing technique developed by Pacific Biosciences. The complete and finished genome sequence of the strain consists of a 4,696,984 bp chromosome featuring a GC-content of 62.34%. Comparative analyses between the new and previous versions of the P. pseudoalcaligenes CECT5344 genome sequence revealed additional regions in the new sequence that were missed in the older version. These additional regions mostly represent mobile genetic elements. Moreover, five additional genes predicted to play a role in sulfoxide reduction are present in the newly established genome sequence. The P. pseudoalcaligenes CECT5344 genome sequence is highly related to the genome sequences of different Pseudomonas mendocina strains. Approximately, 70% of all genes are shared between P. pseudoalcaligenes and P. mendocina. In contrast to P. mendocina, putative pathogenicity genes were not identified in the P. pseudoalcaligenes CECT5344 genome. P. pseudoalcaligenes CECT5344 possesses unique genes for nitrilases and mercury resistance proteins that are of importance for survival in habitats contaminated with cyano- and mercury compounds. As an additional feature of the SMRT sequencing technology, the methylome of P. pseudoalcaligenes was established. Six sequence motifs featuring methylated adenine residues (m6A) were identified in the genome. The genome encodes several methyltransferases, some of which may be considered for methylation of the m6A motifs identified. The complete genome sequence of the strain CECT5344 now provides the basis for exploitation of genetic features for biotechnological purposes. Copyright © 2016 Elsevier B.V. All rights reserved.

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July 7, 2019  |  

Complete genome sequence of Pseudoalteromonas rubra SCSIO 6842, harboring a putative conjugative plasmid pMBL6842.

Pseudoalteromonas is a genus of Gram-negative and is ubiquitously distributed in the ocean. Many Pseudoalteromonas species are capable of producing pigments, which can serve as an alternative source to replace synthetic pigments used in the food industry. Prodigiosins belong to a family of secondary metabolite characterized by a common pyrrolyl pyrromethane skeleton, and have been successfully applied to yogurt, milk and carbonated drinks as substitutes for synthetic additives. The strain Pseudoalteromonas rubra SCSIO 6842 can produce cycloprodigiosin and harbors a conjugative plasmid. Here we report the complete genome of P. rubra SCSIO 6842 for a better understanding of the molecular basis of cycloprodigiosin production and regulation. Copyright © 2016 Elsevier B.V. All rights reserved.

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July 7, 2019  |  

Genome sequence of Aeromicrobium erythreum NRRL B-3381, an erythromycin-producing bacterium of the Nocardioidaceae.

Aeromicrobium erythreumNRRL B-3381 has a 3,629,239-bp circular genome that has 72% G+C content. There are at least 3,121 coding sequences (CDSs), two rRNA gene operons, and 47 tRNAs. The genome and erythromycin (ery) biosynthetic gene sequences provide resources for metabolic and combinatorial engineering of polyketides. Copyright © 2016 Harrell and Miller.

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July 7, 2019  |  

Next-generation sequencing-based detection of germline L1-mediated transductions.

While active LINE-1 (L1) elements possess the ability to mobilize flanking sequences to different genomic loci through a process termed transduction influencing genomic content and structure, an approach for detecting polymorphic germline non-reference transductions in massively-parallel sequencing data has been lacking.Here we present the computational approach TIGER (Transduction Inference in GERmline genomes), enabling the discovery of non-reference L1-mediated transductions by combining L1 discovery with detection of unique insertion sequences and detailed characterization of insertion sites. We employed TIGER to characterize polymorphic transductions in fifteen genomes from non-human primate species (chimpanzee, orangutan and rhesus macaque), as well as in a human genome. We achieved high accuracy as confirmed by PCR and two single molecule DNA sequencing techniques, and uncovered differences in relative rates of transduction between primate species.By enabling detection of polymorphic transductions, TIGER makes this form of relevant structural variation amenable for population and personal genome analysis.

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July 7, 2019  |  

Assembly of long error-prone reads using de Bruijn graphs.

The recent breakthroughs in assembling long error-prone reads were based on the overlap-layout-consensus (OLC) approach and did not utilize the strengths of the alternative de Bruijn graph approach to genome assembly. Moreover, these studies often assume that applications of the de Bruijn graph approach are limited to short and accurate reads and that the OLC approach is the only practical paradigm for assembling long error-prone reads. We show how to generalize de Bruijn graphs for assembling long error-prone reads and describe the ABruijn assembler, which combines the de Bruijn graph and the OLC approaches and results in accurate genome reconstructions.

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July 7, 2019  |  

Complete genome sequence of pseudorabies virus reference strain NIA3 using single-molecule real-time sequencing.

Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease in pigs. PRV strains are also used as model organisms for the study of alphaherpesvirus biology or for neuronal pathway studies. We present here the complete genome of the virulent wild-type PRV reference strain NIA3, determined by single-molecule real-time sequencing. Copyright © 2016 Mathijs et al.

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July 7, 2019  |  

Long single-molecule reads can resolve the complexity of the influenza virus composed of rare, closely related mutant variants

As a result of a high rate of mutations and recombination events, an RNA-virus exists as a heterogeneous “swarm” of mutant variants. The long read length offered by single-molecule sequencing technologies allows each mutant variant to be sequenced in a single pass. However, high error rate limits the ability to reconstruct heterogeneous viral population composed of rare, related mutant variants. In this paper, we present 2SNV, a method able to tolerate the high error-rate of the single-molecule protocol and reconstruct mutant variants. 2SNV uses linkage between single nucleotide variations to efficiently distinguish them from read errors. To benchmark the sensitivity of 2SNV, we performed a single-molecule sequencing experiment on a sample containing a titrated level of known viral mutant variants. Our method is able to accurately reconstruct clone with frequency of 0.2 % and distinguish clones that differed in only two nucleotides distantly located on the genome. 2SNV outperforms existing methods for full-length viral mutant reconstruction. The open source implementation of 2SNV is freely available for download at http://?alan.?cs.?gsu.?edu/?NGS/???q=?content/?2snv.

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July 7, 2019  |  

Complete genome sequence of Wohlfahrtiimonas chitiniclastica strain BM-Y, isolated from the pancreas of a zebra in China.

Here, a complete genome sequence of Wohlfahrtiimonas chitiniclastica strain BM-Y is presented. The whole genome is 2.18-Mb and contains a blaVEB-1 gene cassette which endows it with resistance to ceftazidime, ampicillin, tetracycline, etc. To our knowledge, this is the first time that an extended spectrum beta-lactamase (ESBL) type W. chitiniclastica strain has been found. Copyright © 2016 Zhou et al.

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July 7, 2019  |  

Genomics-informed isolation and characterization of a symbiotic Nanoarchaeota system from a terrestrial geothermal environment.

Biological features can be inferred, based on genomic data, for many microbial lineages that remain uncultured. However, cultivation is important for characterizing an organism’s physiology and testing its genome-encoded potential. Here we use single-cell genomics to infer cultivation conditions for the isolation of an ectosymbiotic Nanoarchaeota (‘Nanopusillus acidilobi’) and its host (Acidilobus, a crenarchaeote) from a terrestrial geothermal environment. The cells of ‘Nanopusillus’ are among the smallest known cellular organisms (100-300?nm). They appear to have a complete genetic information processing machinery, but lack almost all primary biosynthetic functions as well as respiration and ATP synthesis. Genomic and proteomic comparison with its distant relative, the marine Nanoarchaeum equitans illustrate an ancient, common evolutionary history of adaptation of the Nanoarchaeota to ectosymbiosis, so far unique among the Archaea.

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July 7, 2019  |  

Complete genome sequence of highly virulent Haemophilus parasuis serotype 11 strain SC1401.

Haemophilus parasuis, a normal Gram-negative bacterium, may cause Glässer’s disease and pneumonia in pigs. This study aims to identify the genes related to natural competence of the serotype 11 strain SC1401, which frequently shows competence and high pathogenicity. SC1401 shows many differences from strains without natural competence within the molecular basis. We performed complete genome sequencing together with restriction modification system analysis to lay the foundation for later study. Copyright © 2016 Dai et al.

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