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July 7, 2019

Current status of genome sequencing and its applications in aquaculture

Aquaculture is the fastest-growing food production sector in agriculture, with great potential to meet projected protein needs of human beings. Aquaculture is facing several challenges, including lack of a sufficient number of genetically improved species, lack of species-specific feeds, high mortality due to diseases and pollution of ecosystems. The rapid development of sequencing technologies has revolutionized biological sciences, and supplied necessary tools to tackle these challenges in aquaculture and thus ensure its sustainability and profitability. So far, draft genomes have been published in over 24 aquaculture species, and used to address important issues related to aquaculture. We briefly review the advances of next generation sequencing technologies, and summarize the status of whole genome sequencing and its general applications (i.e. establishing reference genomes and discovering DNA markers) and specific applications in tackling some important issues (e.g. breeding, diseases, sex determination & maturation) related to aquaculture. For sequencing a new genome, we recommend the use of 100–200 × short reads using Illumina and 50–60 × long reads with PacBio sequencing technologies. For identification of a large number of SNPs, resequencing pooled DNA samples from different populations is the most cost-effective way. We also discuss the challenges and future directions of whole genome sequencing in aquaculture.

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July 7, 2019

Strategies for complete plastid genome sequencing.

Plastid sequencing is an essential tool in the study of plant evolution. This high-copy organelle is one of the most technically accessible regions of the genome, and its sequence conservation makes it a valuable region for comparative genome evolution, phylogenetic analysis and population studies. Here, we discuss recent innovations and approaches for de novo plastid assembly that harness genomic tools. We focus on technical developments including low-cost sequence library preparation approaches for genome skimming, enrichment via hybrid baits and methylation-sensitive capture, sequence platforms with higher read outputs and longer read lengths, and automated tools for assembly. These developments allow for a much more streamlined assembly than via conventional short-range PCR. Although newer methods make complete plastid sequencing possible for any land plant or green alga, there are still challenges for producing finished plastomes particularly from herbarium material or from structurally divergent plastids such as those of parasitic plants.© 2016 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

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July 7, 2019

NOVOPlasty: de novo assembly of organelle genomes from whole genome data.

The evolution in next-generation sequencing (NGS) technology has led to the development of many different assembly algorithms, but few of them focus on assembling the organelle genomes. These genomes are used in phylogenetic studies, food identification and are the most deposited eukaryotic genomes in GenBank. Producing organelle genome assembly from whole genome sequencing (WGS) data would be the most accurate and least laborious approach, but a tool specifically designed for this task is lacking. We developed a seed-and-extend algorithm that assembles organelle genomes from whole genome sequencing (WGS) data, starting from a related or distant single seed sequence. The algorithm has been tested on several new (Gonioctena intermedia and Avicennia marina) and public (Arabidopsis thaliana and Oryza sativa) whole genome Illumina data sets where it outperforms known assemblers in assembly accuracy and coverage. In our benchmark, NOVOPlasty assembled all tested circular genomes in less than 30 min with a maximum memory requirement of 16 GB and an accuracy over 99.99%. In conclusion, NOVOPlasty is the sole de novo assembler that provides a fast and straightforward extraction of the extranuclear genomes from WGS data in one circular high quality contig. The software is open source and can be downloaded at https://github.com/ndierckx/NOVOPlasty.

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July 7, 2019

Prevalence and molecular characterization of mcr-1-positive Salmonella strains recovered from clinical specimens in China.

The recently discovered colistin resistance element, mcr-1, adds to the list of antimicrobial resistance genes that rapidly erode the antimicrobial efficacy of not only the commonly used antibiotics but also the last-line agents of carbapenems and colistin. This study investigated the prevalence of the mobile colistin resistance determinant mcr-1 in Salmonella strains recovered from clinical settings in China and the transmission potential of mcr-1-bearing mobile elements harbored by such isolates. The mcr-1 gene was recoverable in 1.4% of clinical isolates tested, with the majority of them belonging to Salmonella enterica serotype Typhimurium. These isolates exhibited diverse pulsed-field gel electrophoresis (PFGE) profiles and high resistance to antibiotics other than colistin and particularly to cephalosporins. Plasmid analysis showed that mcr-1 was carried on a variety of plasmids with sizes ranging from ~30 to ~250 kb, among which there were conjugative plasmids of ~30 kb, ~60 kb, and ~250 kb and nonconjugative plasmids of ~140 kb, ~180 kb, and ~240 kb. Sequencing of representative mcr-1-carrying plasmids revealed that all conjugative plasmids belonged to the IncX4, IncI2, and IncHI2 types and were highly similar to the corresponding types of plasmids reported previously. Nonconjugative plasmids all belonged to the IncHI2 type, and the nontransferability of these plasmids was attributed to the loss of a region carrying partial or complete tra genes. Our data revealed that, similar to the situation in Escherichia coli, mcr-1 transmission in Salmonella was accelerated by various plasmids, suggesting that transmission of mcr-1-carrying plasmids between different species of Enterobacteriaceae may be a common event. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Mistranslation can enhance fitness through purging of deleterious mutations.

Phenotypic mutations are amino acid changes caused by mistranslation. How phenotypic mutations affect the adaptive evolution of new protein functions is unknown. Here we evolve the antibiotic resistance protein TEM-1 towards resistance on the antibiotic cefotaxime in an Escherichia coli strain with a high mistranslation rate. TEM-1 populations evolved in such strains endow host cells with a general growth advantage, not only on cefotaxime but also on several other antibiotics that ancestral TEM-1 had been unable to deactivate. High-throughput sequencing of TEM-1 populations shows that this advantage is associated with a lower incidence of weakly deleterious genotypic mutations. Our observations show that mistranslation is not just a source of noise that delays adaptive evolution. It could even facilitate adaptive evolution by exacerbating the effects of deleterious mutations and leading to their more efficient purging. The ubiquity of mistranslation and its effects render mistranslation an important factor in adaptive protein evolution.

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July 7, 2019

Complete genome of a panresistant Pseudomonas aeruginosa strain, isolated from a patient with respiratory failure in a Canadian community hospital.

We report here the complete genome sequence of a panresistant Pseudomonas aeruginosa strain, isolated from a patient with respiratory failure in Canada. No carbapenemase genes were identified. Carbapenem resistance is attributable to a frameshift in the oprD gene; the basis for colistin resistance remains undetermined. Copyright © 2017 Xiong et al.

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July 7, 2019

Evolutionary origin of the staphylococcal cassette chromosome mec (SCCmec).

Several lines of evidence indicate that the most primitive staphylococcal species, those of the Staphylococcus sciuri group, were involved in the first stages of evolution of the staphylococcal cassette chromosome mec (SCCmec), the genetic element carrying the ß-lactam resistance gene mecA However, many steps are still missing from this evolutionary history. In particular, it is not known how mecA was incorporated into the mobile element SCC prior to dissemination among Staphylococcus aureus and other pathogenic staphylococcal species. To gain insights into the possible contribution of several species of the Staphylococcus sciuri group to the assembly of SCCmec, we sequenced the genomes of 106 isolates, comprising S. sciuri (n = 76), Staphylococcus vitulinus (n = 18), and Staphylococcus fleurettii (n = 12) from animal and human sources, and characterized the native location of mecA and the SCC insertion site by using a variety of comparative genomic approaches. Moreover, we performed a single nucleotide polymorphism (SNP) analysis of the genomes in order to understand SCCmec evolution in relation to phylogeny. We found that each of three species of the S. sciuri group contributed to the evolution of SCCmec: S. vitulinus and S. fleurettii contributed to the assembly of the mec complex, and S. sciuri most likely provided the mobile element in which mecA was later incorporated. We hypothesize that an ancestral SCCmec III cassette (an element carried by one of the most epidemic methicillin-resistant S. aureus clones) originated in S. sciuri possibly by a recombination event in a human host or a human-created environment and later was transferred to S. aureus. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

MHC class I diversity in chimpanzees and bonobos.

Major histocompatibility complex (MHC) class I genes are critically involved in the defense against intracellular pathogens. MHC diversity comparisons among samples of closely related taxa may reveal traces of past or ongoing selective processes. The bonobo and chimpanzee are the closest living evolutionary relatives of humans and last shared a common ancestor some 1 mya. However, little is known concerning MHC class I diversity in bonobos or in central chimpanzees, the most numerous and genetically diverse chimpanzee subspecies. Here, we used a long-read sequencing technology (PacBio) to sequence the classical MHC class I genes A, B, C, and A-like in 20 and 30 wild-born bonobos and chimpanzees, respectively, with a main focus on central chimpanzees to assess and compare diversity in those two species. We describe in total 21 and 42 novel coding region sequences for the two species, respectively. In addition, we found evidence for a reduced MHC class I diversity in bonobos as compared to central chimpanzees as well as to western chimpanzees and humans. The reduced bonobo MHC class I diversity may be the result of a selective process in their evolutionary past since their split from chimpanzees.

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July 7, 2019

Designing robust watermark barcodes for multiplex long-read sequencing.

To attain acceptable sample misassignment rates, current approaches to multiplex single-molecule real-time sequencing require upstream quality improvement, which is obtained from multiple passes over the sequenced insert and significantly reduces the effective read length. In order to fully exploit the raw read length on multiplex applications, robust barcodes capable of dealing with the full single-pass error rates are needed.We present a method for designing sequencing barcodes that can withstand a large number of insertion, deletion and substitution errors and are suitable for use in multiplex single-molecule real-time sequencing. The manuscript focuses on the design of barcodes for full-length single-pass reads, impaired by challenging error rates in the order of 11%. The proposed barcodes can multiplex hundreds or thousands of samples while achieving sample misassignment probabilities as low as 10-7 under the above conditions, and are designed to be compatible with chemical constraints imposed by the sequencing process.Software tools for constructing watermark barcode sets and demultiplexing barcoded reads, together with example sets of barcodes and synthetic barcoded reads, are freely available at www.cifasis-conicet.gov.ar/ezpeleta/NS-watermark .ezpeleta@cifasis-conicet.gov.ar.

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July 7, 2019

Geographical structure of endosymbiotic bacteria hosted by Bathymodiolus mussels at eastern Pacific hydrothermal vents.

Chemolithoautotrophic primary production sustains dense invertebrate communities at deep-sea hydrothermal vents and hydrocarbon seeps. Symbiotic bacteria that oxidize dissolved sulfur, methane, and hydrogen gases nourish bathymodiolin mussels that thrive in these environments worldwide. The mussel symbionts are newly acquired in each generation via infection by free-living forms. This study examined geographical subdivision of the thiotrophic endosymbionts hosted by Bathymodiolus mussels living along the eastern Pacific hydrothermal vents. High-throughput sequencing data of 16S ribosomal RNA encoding gene and fragments of six protein-coding genes of symbionts were examined in the samples collected from nine vent localities at the East Pacific Rise, Galápagos Rift, and Pacific-Antarctic Ridge.Both of the parapatric sister-species, B. thermophilus and B. antarcticus, hosted the same numerically dominant phylotype of thiotrophic Gammaproteobacteria. However, sequences from six protein-coding genes revealed highly divergent symbiont lineages living north and south of the Easter Microplate and hosted by these two Bathymodiolus mussel species. High heterogeneity of symbiont haplotypes among host individuals sampled from the same location suggested that stochasticity associated with initial infections was amplified as symbionts proliferated within the host individuals. The mussel species presently contact one another and hybridize along the Easter Microplate, but the northern and southern symbionts appear to be completely isolated. Vicariance associated with orogeny of the Easter Microplate region, 2.5-5.3 million years ago, may have initiated isolation of the symbiont and host populations. Estimates of synonymous substitution rates for the protein-coding bacterial genes examined in this study were 0.77-1.62%/nucleotide/million years.Our present study reports the most comprehensive population genetic analyses of the chemosynthetic endosymbiotic bacteria based on high-throughput genetic data and extensive geographical sampling to date, and demonstrates the role of the geographical features, the Easter Microplate and geographical distance, in the intraspecific divergence of this bacterial species along the mid-ocean ridge axes in the eastern Pacific. Altogether, our results provide insights into extrinsic and intrinsic factors affecting the dispersal and evolution of chemosynthetic symbiotic partners in the hydrothermal vents along the eastern Pacific Ocean.

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July 7, 2019

A novel inversion in the chloroplast genome of marama (Tylosema esculentum).

Tylosema esculentum (marama bean) is being developed as a possible crop for resource-poor farmers in arid regions of Southern Africa. As part of the molecular characterization of this species, the chloroplast genome has been assembled from next-generation sequencing using both Illumina and Pac-Bio data. The genome is of typical organization with a large single-copy region and a small single-copy region separated by a pair of inverted repeats and covers 161537 bp. It contains a unique inversion not present in any other legumes, even in the closest relatives for which the complete chloroplast genome is available, and two complete copies of the ycf1 gene. These data extend the range of variability of legume chloroplast genomes. The sequencing of multiple individuals has identified two different chloroplast genomes which were geographically separated. The current sampling is limited so that the extent of the intraspecific variation is still to be determined, leaving open the question of legume chloroplast genomes adapted to particular arid environments.© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

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July 7, 2019

Whole-genome restriction mapping by “subhaploid”-based RAD sequencing: An efficient and flexible approach for physical mapping and genome scaffolding.

Assembly of complex genomes using short reads remains a major challenge, which usually yields highly fragmented assemblies. Generation of ultradense linkage maps is promising for anchoring such assemblies, but traditional linkage mapping methods are hindered by the infrequency and unevenness of meiotic recombination that limit attainable map resolution. Here we develop a sequencing-based “in vitro” linkage mapping approach (called RadMap), where chromosome breakage and segregation are realized by generating hundreds of “subhaploid” fosmid/bacterial-artificial-chromosome clone pools, and by restriction site-associated DNA sequencing of these clone pools to produce an ultradense whole-genome restriction map to facilitate genome scaffolding. A bootstrap-based minimum spanning tree algorithm is developed for grouping and ordering of genome-wide markers and is implemented in a user-friendly, integrated software package (AMMO). We perform extensive analyses to validate the power and accuracy of our approach in the model plant Arabidopsis thaliana and human. We also demonstrate the utility of RadMap for enhancing the contiguity of a variety of whole-genome shotgun assemblies generated using either short Illumina reads (300 bp) or long PacBio reads (6-14 kb), with up to 15-fold improvement of N50 (~816 kb-3.7 Mb) and high scaffolding accuracy (98.1-98.5%). RadMap outperforms BioNano and Hi-C when input assembly is highly fragmented (contig N50 = 54 kb). RadMap can capture wide-range contiguity information and provide an efficient and flexible tool for high-resolution physical mapping and scaffolding of highly fragmented assemblies. Copyright © 2017 Dou et al.

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July 7, 2019

Phylogenomic analysis supports multiple instances of polyphyly in the oomycete peronosporalean lineage.

The study of biological diversification of oomycetes has been a difficult task for more than a century. Pioneer researchers used morphological characters to describe this heterogeneous group, and physiological and genetic tools expanded knowledge of these microorganisms. However, research on oomycete diversification is limited by conflicting phylogenies. Using whole genomic data from 17 oomycete taxa, we obtained a dataset of 277 core orthologous genes shared among these genomes. Analyses of this dataset resulted in highly congruent and strongly supported estimates of oomycete phylogeny when we used concatenated maximum likelihood and coalescent-based methods; the one important exception was the position of Albugo. Our results supported the position of Phytopythium vexans (formerly in Pythium clade K) as a sister clade to the Phytophthora-Hyaloperonospora clade. The remaining clades comprising Pythium sensu lato formed two monophyletic groups. One group was composed of three taxa that correspond to Pythium clades A, B and C, and the other group contained taxa representing clades F, G and I, in agreement with previous Pythium phylogenies. However, the group containing Pythium clades F, G and I was placed as sister to the Phytophthora-Hyaloperonospora-Phytopythium clade, thus confirming the lack of monophyly of Pythium sensu lato. Multispecies coalescent methods revealed that the white blister rust, Albugo laibachii, could not be placed with a high degree of confidence. Our analyses show that genomic data can resolve the oomycete phylogeny and provide a phylogenetic framework to study the evolution of oomycete lifestyles. Copyright © 2017 Elsevier Inc. All rights reserved.

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July 7, 2019

PipeCraft: Flexible open-source toolkit for bioinformatics analysis of custom high-throughput amplicon sequencing data.

High-throughput sequencing methods have become a routine analysis tool in environmental sciences as well as in public and private sector. These methods provide vast amount of data, which need to be analysed in several steps. Although the bioinformatics may be applied using several public tools, many analytical pipelines allow too few options for the optimal analysis for more complicated or customized designs. Here, we introduce PipeCraft, a flexible and handy bioinformatics pipeline with a user-friendly graphical interface that links several public tools for analysing amplicon sequencing data. Users are able to customize the pipeline by selecting the most suitable tools and options to process raw sequences from Illumina, Pacific Biosciences, Ion Torrent and Roche 454 sequencing platforms. We described the design and options of PipeCraft and evaluated its performance by analysing the data sets from three different sequencing platforms. We demonstrated that PipeCraft is able to process large data sets within 24 hr. The graphical user interface and the automated links between various bioinformatics tools enable easy customization of the workflow. All analytical steps and options are recorded in log files and are easily traceable.© 2017 John Wiley & Sons Ltd.

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July 7, 2019

Phylogeography of Burkholderia pseudomallei isolates, Western Hemisphere.

The bacterium Burkholderia pseudomallei causes melioidosis, which is mainly associated with tropical areas. We analyzed single-nucleotide polymorphisms (SNPs) among genome sequences from isolates of B. pseudomallei that originated in the Western Hemisphere by comparing them with genome sequences of isolates that originated in the Eastern Hemisphere. Analysis indicated that isolates from the Western Hemisphere form a distinct clade, which supports the hypothesis that these isolates were derived from a constricted seeding event from Africa. Subclades have been resolved that are associated with specific regions within the Western Hemisphere and suggest that isolates might be correlated geographically with cases of melioidosis. One isolate associated with a former World War II prisoner of war was believed to represent illness 62 years after exposure in Southeast Asia. However, analysis suggested the isolate originated in Central or South America.

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