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Asset Tag: single molecule real time

July 7, 2019  |  

Genome puzzle master (GPM): an integrated pipeline for building and editing pseudomolecules from fragmented sequences.

Next generation sequencing technologies have revolutionized our ability to rapidly and affordably generate vast quantities of sequence data. Once generated, raw sequences are assembled into contigs or scaffolds. However, these assemblies are mostly fragmented and inaccurate at the whole genome scale, largely due to the inability to integrate additional informative datasets (e.g. physical, optical and genetic maps). To address this problem, we developed a semi-automated software tool-Genome Puzzle Master (GPM)-that enables the integration of additional genomic signposts to edit and build ‘new-gen-assemblies’ that result in high-quality ‘annotation-ready’ pseudomolecules.With GPM, loaded datasets can be connected to each other via their logical relationships which accomplishes tasks to ‘group,’ ‘merge,’ ‘order and orient’ sequences in a draft assembly. Manual editing can also be performed with a user-friendly graphical interface. Final pseudomolecules reflect a user’s total data package and are available for long-term project management. GPM is a web-based pipeline and an important part of a Laboratory Information Management System (LIMS) which can be easily deployed on local servers for any genome research laboratory.The GPM (with LIMS) package is available at https://github.com/Jianwei-Zhang/LIMS CONTACTS: jzhang@mail.hzau.edu.cn or rwing@mail.arizona.eduSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

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July 7, 2019  |  

First complete genome sequence of the skin-improving Lactobacillus curvatus strain FBA2, isolated from fermented vegetables, determined by PacBio single-molecule real-time technology.

The first complete genome sequence of Lactobacillus curvatus was determined by PacBio RS II. The single circular chromosome (1,848,756 bp, G+C content of 42.1%) of L. curvatus FBA2, isolated from fermented vegetables, contained low G+C regions (26.9% minimum) and 43 sets of >1,000-bp identical sequence pairs. No plasmids were detected. Copyright © 2016 Nakano et al.

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July 7, 2019  |  

Draft genome sequence of Mycobacterium rufum JS14(T), a polycyclic-aromatic-hydrocarbon-degrading bacterium from petroleum-contaminated soil in Hawaii.

Mycobacterium rufum JS14(T) (=ATCC BAA-1377(T), CIP 109273(T), JCM 16372(T), DSM 45406(T)), a type strain of the species Mycobacterium rufum sp. . belonging to the family Mycobacteriaceae, was isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soil in Hilo (HI, USA) because it harbors the capability of degrading PAH. Here, we describe the first genome sequence of strain JS14(T), with brief phenotypic characteristics. The genome is composed of 6,176,413 bp with 69.25 % G?+?C content and contains 5810 protein-coding genes with 54 RNA genes. The genome information on M. rufum JS14(T) will provide a better understanding of the complexity of bacterial catabolic pathways for degradation of specific chemicals.

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July 7, 2019  |  

The report of my death was an exaggeration: A review for researchers using microsatellites in the 21st century.

Microsatellites, or simple sequence repeats (SSRs), have long played a major role in genetic studies due to their typically high polymorphism. They have diverse applications, including genome mapping, forensics, ascertaining parentage, population and conservation genetics, identification of the parentage of polyploids, and phylogeography. We compare SSRs and newer methods, such as genotyping by sequencing (GBS) and restriction site associated DNA sequencing (RAD-Seq), and offer recommendations for researchers considering which genetic markers to use. We also review the variety of techniques currently used for identifying microsatellite loci and developing primers, with a particular focus on those that make use of next-generation sequencing (NGS). Additionally, we review software for microsatellite development and report on an experiment to assess the utility of currently available software for SSR development. Finally, we discuss the future of microsatellites and make recommendations for researchers preparing to use microsatellites. We argue that microsatellites still have an important place in the genomic age as they remain effective and cost-efficient markers.

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July 7, 2019  |  

High quality draft genome sequence of the type strain of Pseudomonas lutea OK2(T), a phosphate-solubilizing rhizospheric bacterium.

Pseudomonas lutea OK2(T) (=LMG 21974(T), CECT 5822(T)) is the type strain of the species and was isolated from the rhizosphere of grass growing in Spain in 2003 based on its phosphate-solubilizing capacity. In order to identify the functional significance of phosphate solubilization in Pseudomonas Plant growth promoting rhizobacteria, we describe here the phenotypic characteristics of strain OK2(T) along with its high-quality draft genome sequence, its annotation, and analysis. The genome is comprised of 5,647,497 bp with 60.15 % G?+?C content. The sequence includes 4,846 protein-coding genes and 95 RNA genes.

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July 7, 2019  |  

Improved hybrid de novo genome assembly of domesticated apple (Malus x domestica).

Domesticated apple (Malus?×?domestica Borkh) is a popular temperate fruit with high nutrient levels and diverse flavors. In 2012, global apple production accounted for at least one tenth of all harvested fruits. A high-quality apple genome assembly is crucial for the selection and breeding of new cultivars. Currently, a single reference genome is available for apple, assembled from 16.9?×?genome coverage short reads via Sanger and 454 sequencing technologies. Although a useful resource, this assembly covers only ~89 % of the non-repetitive portion of the genome, and has a relatively short (16.7 kb) contig N50 length. These downsides make it difficult to apply this reference in transcriptive or whole-genome re-sequencing analyses.Here we present an improved hybrid de novo genomic assembly of apple (Golden Delicious), which was obtained from 76 Gb (~102?×?genome coverage) Illumina HiSeq data and 21.7 Gb (~29?×?genome coverage) PacBio data. The final draft genome is approximately 632.4 Mb, representing?~?90 % of the estimated genome. The contig N50 size is 111,619 bp, representing a 7 fold improvement. Further annotation analyses predicted 53,922 protein-coding genes and 2,765 non-coding RNA genes.The new apple genome assembly will serve as a valuable resource for investigating complex apple traits at the genomic level. It is not only suitable for genome editing and gene cloning, but also for RNA-seq and whole-genome re-sequencing studies.

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July 7, 2019  |  

Isolation and genomic characterization of ‘Desulfuromonas soudanensis WTL’, a metal- and electrode-respiring bacterium from anoxic deep subsurface brine.

Reaching a depth of 713 m below the surface, the Soudan Underground Iron Mine (Soudan, MN, USA) transects a massive Archaean (2.7 Ga) banded iron formation, providing a remarkably accessible window into the terrestrial deep biosphere. Despite organic carbon limitation, metal-reducing microbial communities are present in potentially ancient anoxic brines continuously emanating from exploratory boreholes on Level 27. Using graphite electrodes deposited in situ as bait, we electrochemically enriched and isolated a novel halophilic iron-reducing Deltaproteobacterium, ‘Desulfuromonas soudanensis’ strain WTL, from an acetate-fed three-electrode bioreactor poised at +0.24 V (vs. standard hydrogen electrode). Cyclic voltammetry revealed that ‘D. soudanensis’ releases electrons at redox potentials approximately 100 mV more positive than the model freshwater surface isolate Geobacter sulfurreducens, suggesting that its extracellular respiration is tuned for higher potential electron acceptors. ‘D. soudanensis’ contains a 3,958,620-bp circular genome, assembled to completion using single-molecule real-time (SMRT) sequencing reads, which encodes a complete TCA cycle, 38 putative multiheme c-type cytochromes, one of which contains 69 heme-binding motifs, and a LuxI/LuxR quorum sensing cassette that produces an unidentified N-acyl homoserine lactone. Another cytochrome is predicted to lie within a putative prophage, suggesting that horizontal gene transfer plays a role in respiratory flexibility among metal reducers. Isolation of ‘D. soudanensis’ underscores the utility of electrode-based approaches for enriching rare metal reducers from a wide range of habitats.

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July 7, 2019  |  

Complete genome of the starch-degrading myxobacteria Sandaracinus amylolyticus DSM 53668T.

Myxobacteria are members of d-proteobacteria and are typified by large genomes, well-coordinated social behavior, gliding motility, and starvation-induced fruiting body formation. Here, we report the 10.33 Mb whole genome of a starch-degrading myxobacterium Sandaracinus amylolyticus DSM 53668(T) that encodes 8,962 proteins, 56 tRNA, and two rRNA operons. Phylogenetic analysis, in silico DNA-DNA hybridization and average nucleotide identity reveal its divergence from other myxobacterial species and support its taxonomic characterization into a separate family Sandaracinaceae, within the suborder Sorangiineae. Sequence similarity searches using the Carbohydrate-active enzymes (CAZyme) database help identify the enzyme repertoire of S. amylolyticus involved in starch, agar, chitin, and cellulose degradation. We identified 16 a-amylases and two ?-amylases in the S. amylolyticus genome that likely play a role in starch degradation. While many of the amylases are seen conserved in other d-proteobacteria, we notice several novel amylases acquired via horizontal transfer from members belonging to phylum Deinococcus-Thermus, Acidobacteria, and Cyanobacteria. No agar degrading enzyme(s) were identified in the S. amylolyticus genome. Interestingly, several putative ß-glucosidases and endoglucanases proteins involved in cellulose degradation were identified. However, the absence of cellobiohydrolases/exoglucanases corroborates with the lack of cellulose degradation by this bacteria. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 7, 2019  |  

Genome sequence and analysis of Peptoclostridium difficile strain ZJCDC-S82.

Peptoclostridium difficile (Clostridium difficile) is the major pathogen associated with infectious diarrhea in humans. Concomitant with the increased incidence of C. difficile infection worldwide, there is an increasing concern regarding this infection type. This study reports a draft assembly and detailed sequence analysis of C. difficile strain ZJCDC-S82. The de novo assembled genome was 4.19 Mb in size, which includes 4,013 protein-coding genes, 41 rRNA genes, and 84 tRNA genes. Along with the nuclear genome, we also assembled sequencing information for a single plasmid consisting of 11,930 nucleotides. Comparative genomic analysis of C. difficile ZJCDC-S82 and two other previously published strains, such as M120 and CD630, showed extensive similarity. Phylogenetic analysis revealed that genetic diversity among C. difficile strains was not influenced by geographic location. Evolutionary analysis suggested that four genes encoding surface proteins exhibited positive selection in C. difficile ZJCDC-S82. Codon usage analysis indicated that C. difficile ZJCDC-S82 had high codon usage bias toward A/U-ended codons. Furthermore, codon usage patterns in C. difficile ZJCDC-S82 were predominantly affected by mutation pressure. Our results provide detailed information pertaining to the C. difficile genome associated with a strain from mainland China. This analysis will facilitate the understanding of genomic diversity and evolution of C. difficile strains in this region.

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July 7, 2019  |  

Improved complete genome sequence of the extremely radioresistant bacterium Deinococcus radiodurans R1 obtained using PacBio single-molecule sequencing.

The genome sequence of Deinococcus radiodurans R1 was published in 1999. We resequenced D. radiodurans R1 using PacBio and compared the sequence with the published one. Large insertions and single nucleotide polymorphisms (SNPs) were observed among the genome sequences. A more accurate genome sequence will be helpful to studies of D. radiodurans. Copyright © 2016 Hua and Hua.

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July 7, 2019  |  

An ultra-high density genetic linkage map of perennial ryegrass (Lolium perenne) using genotyping by sequencing (GBS) based on a reference shotgun genome assembly.

High density genetic linkage maps that are extensively anchored to assembled genome sequences of the organism in question are extremely useful in gene discovery. To facilitate this process in perennial ryegrass (Lolium perenne L.), a high density single nucleotide polymorphism (SNP)- and presence/absence variant (PAV)-based genetic linkage map has been developed in an F2 mapping population that has been used as a reference population in numerous studies. To provide a reference sequence to which to align genotyping by sequencing (GBS) reads, a shotgun assembly of one of the grandparents of the population, a tenth-generation inbred line, was created using Illumina-based sequencing.The assembly was based on paired-end Illumina reads, scaffolded by mate pair and long jumping distance reads in the range of 3-40?kb, with >200-fold initial genome coverage. A total of 169 individuals from an F2 mapping population were used to construct PstI-based GBS libraries tagged with unique 4-9 nucleotide barcodes, resulting in 284 million reads, with approx. 1·6 million reads per individual. A bioinformatics pipeline was employed to identify both SNPs and PAVs. A core genetic map was generated using high confidence SNPs, to which lower confidence SNPs and PAVs were subsequently fitted in a straightforward binning approach.The assembly comprises 424?750 scaffolds, covering 1·11 Gbp of the 2·5 Gbp perennial ryegrass genome, with a scaffold N50 of 25 212?bp and a contig N50 of 3790?bp. It is available for download, and access to a genome browser has been provided. Comparison of the assembly with available transcript and gene model data sets for perennial ryegrass indicates that approx. 570 Mbp of the gene-rich portion of the genome has been captured. An ultra-high density genetic linkage map with 3092 SNPs and 7260 PAVs was developed, anchoring just over 200?Mb of the reference assembly.The combined genetic map and assembly, combined with another recently released genome assembly, represent a significant resource for the perennial ryegrass genetics community.© The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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July 7, 2019  |  

Assemblytics: a web analytics tool for the detection of variants from an assembly.

Assemblytics is a web app for detecting and analyzing variants from a de novo genome assembly aligned to a reference genome. It incorporates a unique anchor filtering approach to increase robustness to repetitive elements, and identifies six classes of variants based on their distinct alignment signatures. Assemblytics can be applied both to comparing aberrant genomes, such as human cancers, to a reference, or to identify differences between related species. Multiple interactive visualizations enable in-depth explorations of the genomic distributions of variants.http://assemblytics.com, https://github.com/marianattestad/assemblytics CONTACT: mnattest@cshl.eduSupplementary information: Supplementary data are available at Bioinformatics online.© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019  |  

High-quality genome assembly and annotation for Plasmodium coatneyi, generated using single-molecule real-time PacBio technology.

Plasmodium coatneyi is a protozoan parasite species that causes simian malaria and is an excellent model for studying disease caused by the human malaria parasite, P. falciparum Here we report the complete (nontelomeric) genome sequence of P. coatneyi Hackeri generated by the application of only Pacific Biosciences RS II (PacBio RS II) single-molecule real-time (SMRT) high-resolution sequence technology and assembly using the Hierarchical Genome Assembly Process (HGAP). This is the first Plasmodium genome sequence reported to use only PacBio technology. This approach has proven to be superior to short-read only approaches for this species. Copyright © 2016 Chien et al.

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