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April 21, 2020  |  

Complete Genome Sequence of a Chlorobenzene Degrader, Pandoraea pnomenusa MCB032.

Chlorobenzenes are ubiquitously distributed, highly persistent, and toxic environmental contaminants. Pandoraea pnomenusa MCB032 was isolated as a new dominant chlorobenzene-utilizing strain from a functionally stable bioreactor during the treatment of chlorobenzenes when strain Burkholderia sp. JS150 disappeared. In study, we report the complete genome sequence of strain MCB032 which consists of a circular chromosome and three plasmids, which are?~?6 Mb in length with 5450 open reading frames-12 encoding rRNAs and 77 encoding tRNAs. We further identified 17 putative genes encoding the enzymes involved in the methyl-accepting chemotaxis proteins in sensing chemical gradients during chemotaxis. The annotated complete genome sequence of this strain will provide genetic insights into the degradation of chlorinated aromatic compounds. The information will empower the elucidation of chlorobenzene affinity hierarchy and species succession in the bioreactor.


April 21, 2020  |  

De novo assembly of a wild pear (Pyrus betuleafolia) genome.

China is the origin and evolutionary centre of Oriental pears. Pyrus betuleafolia is a wild species native to China and distributed in the northern region, and it is widely used as rootstock. Here, we report the de novo assembly of the genome of P. betuleafolia-Shanxi Duli using an integrated strategy that combines PacBio sequencing, BioNano mapping and chromosome conformation capture (Hi-C) sequencing. The genome assembly size was 532.7 Mb, with a contig N50 of 1.57 Mb. A total of 59 552 protein-coding genes and 247.4 Mb of repetitive sequences were annotated for this genome. The expansion genes in P. betuleafolia were significantly enriched in secondary metabolism, which may account for the organism’s considerable environmental adaptability. An alignment analysis of orthologous genes showed that fruit size, sugar metabolism and transport, and photosynthetic efficiency were positively selected in Oriental pear during domestication. A total of 573 nucleotide-binding site (NBS)-type resistance gene analogues (RGAs) were identified in the P. betuleafolia genome, 150 of which are TIR-NBS-LRR (TNL)-type genes, which represented the greatest number of TNL-type genes among the published Rosaceae genomes and explained the strong disease resistance of this wild species. The study of flavour metabolism-related genes showed that the anthocyanidin reductase (ANR) metabolic pathway affected the astringency of pear fruit and that sorbitol transporter (SOT) transmembrane transport may be the main factor affecting the accumulation of soluble organic matter. This high-quality P. betuleafolia genome provides a valuable resource for the utilization of wild pear in fundamental pear studies and breeding. © 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.


April 21, 2020  |  

The evaluation of RNA-Seq de novo assembly by PacBio long read sequencing

RNA-Seq de novo assembly is an important method to generate transcriptomes for non-model organisms before any downstream analysis. Given many great de novo assembly methods developed by now, one critical issue is that there is no consensus on the evaluation of de novo assembly methods yet. Therefore, to set up a benchmark for evaluating the quality of de novo assemblies is very critical. Addressing this challenge will help us deepen the insights on the properties of different de novo assemblers and their evaluation methods, and provide hints on choosing the best assembly sets as transcriptomes of non-model organisms for the further functional analysis. In this article, we generate a textquotedblleftreal timetextquotedblright transcriptome using PacBio long reads as a benchmark for evaluating five de novo assemblers and two model-based de novo assembly evaluation methods. By comparing the de novo assmblies generated by RNA-Seq short reads with the textquotedblleftreal timetextquotedblright transcriptome from the same biological sample, we find that Trinity is best at the completeness by generating more assemblies than the alternative assemblers, but less continuous and having more misassemblies; Oases is best at the continuity and specificity, but less complete; The performance of SOAPdenovo-Trans, Trans-AByss and IDBA-Tran are in between of five assemblers. For evaluation methods, DETONATE leverages multiple aspects of the assembly set and ranks the assembly set with an average performance as the best, meanwhile the contig score can serve as a good metric to select assemblies with high completeness, specificity, continuity but not sensitive to misassemblies; TransRate contig score is useful for removing misassemblies, yet often the assemblies in the optimal set is too few to be used as a transcriptome.


April 21, 2020  |  

Biochemical characterization of a novel cold-adapted agarotetraose-producing a-agarase, AgaWS5, from Catenovulum sediminis WS1-A.

Although many ß-agarases that hydrolyze the ß-1,4 linkages of agarose have been biochemically characterized, only three a-agarases that hydrolyze the a-1,3 linkages are reported to date. In this study, a new a-agarase, AgaWS5, from Catenovulum sediminis WS1-A, a new agar-degrading marine bacterium, was biochemically characterized. AgaWS5 belongs to the glycoside hydrolase (GH) 96 family. AgaWS5 consists of 1295 amino acids (140 kDa) and has the 65% identity to an a-agarase, AgaA33, obtained from an agar-degrading bacterium Thalassomonas agarivorans JAMB-A33. AgaWS5 showed the maximum activity at a pH and temperature of 8 and 40 °C, respectively. AgaWS5 showed a cold-tolerance, and it retained more than 40% of its maximum enzymatic activity at 10 °C. AgaWS5 is predicted to have several calcium-binding sites. Thus, its activity was slightly enhanced in the presence of Ca2+, and was strongly inhibited by EDTA. The Km and Vmax of AgaWS5 for agarose were 10.6 mg/mL and 714.3 U/mg, respectively. Agarose-liquefication, thin layer chromatography, and mass and NMR spectroscopic analyses demonstrated that AgaWS5 is an endo-type a-agarase that degrades agarose and mainly produces agarotetraose. Thus, in this study, a novel cold-adapted GH96 agarotetraose-producing a-agarase was identified.


April 21, 2020  |  

Chlorella vulgaris genome assembly and annotation reveals the molecular basis for metabolic acclimation to high light conditions.

Chlorella vulgaris is a fast-growing fresh-water microalga cultivated at the industrial scale for applications ranging from food to biofuel production. To advance our understanding of its biology and to establish genetics tools for biotechnological manipulation, we sequenced the nuclear and organelle genomes of Chlorella vulgaris 211/11P by combining next generation sequencing and optical mapping of isolated DNA molecules. This hybrid approach allowed to assemble the nuclear genome in 14 pseudo-molecules with an N50 of 2.8 Mb and 98.9% of scaffolded genome. The integration of RNA-seq data obtained at two different irradiances of growth (high light-HL versus low light -LL) enabled to identify 10,724 nuclear genes, coding for 11,082 transcripts. Moreover 121 and 48 genes were respectively found in the chloroplast and mitochondrial genome. Functional annotation and expression analysis of nuclear, chloroplast and mitochondrial genome sequences revealed peculiar features of Chlorella vulgaris. Evidence of horizontal gene transfers from chloroplast to mitochondrial genome was observed. Furthermore, comparative transcriptomic analyses of LL vs HL provide insights into the molecular basis for metabolic rearrangement in HL vs. LL conditions leading to enhanced de novo fatty acid biosynthesis and triacylglycerol accumulation. The occurrence of a cytosolic fatty acid biosynthetic pathway can be predicted and its upregulation upon HL exposure is observed, consistent with increased lipid amount under HL. These data provide a rich genetic resource for future genome editing studies, and potential targets for biotechnological manipulation of Chlorella vulgaris or other microalgae species to improve biomass and lipid productivity.This article is protected by copyright. All rights reserved.


April 21, 2020  |  

Chromosome-length haplotigs for yak and cattle from trio binning assembly of an F1 hybrid

Background Assemblies of diploid genomes are generally unphased, pseudo-haploid representations that do not correctly reconstruct the two parental haplotypes present in the individual sequenced. Instead, the assembly alternates between parental haplotypes and may contain duplications in regions where the parental haplotypes are sufficiently different. Trio binning is an approach to genome assembly that uses short reads from both parents to classify long reads from the offspring according to maternal or paternal haplotype origin, and is thus helped rather than impeded by heterozygosity. Using this approach, it is possible to derive two assemblies from an individual, accurately representing both parental contributions in their entirety with higher continuity and accuracy than is possible with other methods.Results We used trio binning to assemble reference genomes for two species from a single individual using an interspecies cross of yak (Bos grunniens) and cattle (Bos taurus). The high heterozygosity inherent to interspecies hybrids allowed us to confidently assign >99% of long reads from the F1 offspring to parental bins using unique k-mers from parental short reads. Both the maternal (yak) and paternal (cattle) assemblies contain over one third of the acrocentric chromosomes, including the two largest chromosomes, in single haplotigs.Conclusions These haplotigs are the first vertebrate chromosome arms to be assembled gap-free and fully phased, and the first time assemblies for two species have been created from a single individual. Both assemblies are the most continuous currently available for non-model vertebrates.MbmegabaseskbkilobasesMYAmillions of years agoMHCmajor histocompatibility complexSMRTsingle molecule real time


April 21, 2020  |  

De novo assembly and annotation of the Ganoderma australe genome.

The Ganoderma genus represents clear biotechnological potential, due to the large quantity of molecules with biological activity that could be explored. However, available information regarding the biotechnological importance of species within Ganoderma, other than G. lucidum, is quite limited. Genomic studies of little-known species can contribute to the knowledge thereof, as well as the search for metabolic pathways and the identification of genes which code for proteins that may be of biotechnological relevance. Therefore, the objective of the present study was to obtain the G. australe genome, through the use of new sequencing technologies. Genomic DNA from G. australe was sequenced with the PacBio Sequel system, to a depth of 100×. The genome was assembled de novo with the Canu assembly tool, and gene prediction and annotation were performed with a funannotate pipeline. An assembled 84?Mb genome was obtained, and 22,756 putative protein-coding sequences were predicted in the G. australe genome. Ganoderic acid pathways were annotated and listed in the funannotate pipeline, and were recognized using Pfam and Antismash signals. Thus, the G. australe genome shows great potential, mainly, due to the annotation of putative sequences that could be employed in biotechnological approaches. Copyright © 2019 Elsevier Inc. All rights reserved.


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 one for an outbred, composite breed male animal commonly used for commercial pork production (USMARCv1.0). Both assemblies are of substantially higher (>90-fold) continuity and accuracy compared to the earlier reference, and the availability of two independent assemblies provided an opportunity to identify large-scale variants and to error-check the accuracy of representation of the genome. We propose that the improved Duroc breed assembly (Sscrofa11.1) become the reference genome for genomic research in pigs.


April 21, 2020  |  

Genome-Wide Association Study of Growth and Body-Shape-Related Traits in Large Yellow Croaker (Larimichthys crocea) Using ddRAD Sequencing.

Large yellow croaker (Larimichthys crocea) is an economically important marine fish species of China. Due to overfishing and marine pollution, the wild stocks of this croaker have collapsed in the past decades. Meanwhile, the cultured croaker is facing the difficulties of reduced genetic diversity and low growth rate. To explore the molecular markers related to the growth traits of croaker and providing the related SNPs for the marker-assisted selection, we used double-digest restriction-site associated DNA (ddRAD) sequencing to dissect the genetic bases of growth traits in a cultured population and identify the SNPs that associated with important growth traits by GWAS. A total of 220 individuals were genotyped by ddRAD sequencing. After quality control, 27,227 SNPs were identified in 220 samples and used for GWAS analysis. We identified 13 genome-wide significant associated SNPs of growth traits on 8 chromosomes, and the beta P of these SNPs ranged from 0.01 to 0.86. Through the definition of candidate regions and gene annotation, candidate genes related to growth were identified, including important regulators such as fgf18, fgf1, nr3c1, cyp8b1, fabp2, cyp2r1, ppara, and ccm2l. We also identified SNPs and candidate genes that significantly associated with body shape, including bmp7, col1a1, col11a2, and col18a1, which are also economically important traits for large yellow croaker aquaculture. The results provided insights into the genetic basis of growth and body shape in large yellow croaker population and would provide reliable genetic markers for molecular marker-assisted selection in the future. Meanwhile, the result established a basis for our subsequent fine mapping and related gene study.


April 21, 2020  |  

Resistome and a Novel blaNDM-1-Harboring Plasmid of an Acinetobacter haemolyticus Strain from a Children’s Hospital in Puebla, Mexico.

Acinetobacter calcoaceticus-baumannii complex isolates have been frequently associated with hospital and community infections, with A. baumannii being the most common. Other Acinetobacter spp. not belonging to this complex also cause infections in hospital settings, and the incidence has increased over the past few years. Some species of the Acinetobacter genus possess a great diversity of antibiotic resistance mechanisms, such as efflux pumps, porins, and resistance genes that can be acquired and disseminated by mobilizable genetic elements. By means of whole-genome sequencing, we describe in the clinical Acinetobacter haemolyticus strain AN54 different mechanisms of resistance that involve blaOXA-265, blaNDM-1, aphA6, aac(6′)-Ig, and a resistance-nodulation-cell division-type efflux pump. This strain carries six plasmids, of which the plasmid pAhaeAN54e contains blaNDM-1 in a Tn125-like transposon that is truncated at the 3′ end. This strain also has an insertion sequence IS91 and seven genes encoding hypothetical proteins. The pAhaeAN54e plasmid is nontypable and different from other plasmids carrying blaNDM-1 that have been reported in Mexico and other countries. The presence of these kinds of plasmids in an opportunistic pathogen such as A. haemolyticus highlights the role that these plasmids play in the dissemination of antibiotic resistance genes, especially against carbapenems, in Mexican hospitals.


April 21, 2020  |  

An Outbreak of KPC-Producing Klebsiella pneumoniae Linked with an Index Case of Community-Acquired KPC-Producing Isolate: Epidemiological Investigation and Whole Genome Sequencing Analysis.

Aims: A hospital outbreak of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPN) linked with an index case of community-acquired infection occurred in an urban tertiary care hospital in Seoul, South Korea. Therefore, we performed an outbreak investigation and whole genome sequencing (WGS) analysis to trace the outbreak and investigate the molecular characteristics of the isolates. Results: From October 2014 to January 2015, we identified a cluster of three patients in the neurosurgery ward with sputum cultures positive for carbapenem-resistant KPN. An epidemiological investigation, including pulsed-field gel electrophoresis analysis was performed to trace the origins of this outbreak. The index patient’s infection was community acquired. Active surveillance cultures using perirectal swabbing from exposed patients, identified one additional patient with KPC-producing KPN colonization. WGS analyses using PacBio RSII instruments were performed for four linked isolates. WGS revealed a genetic linkage of the four isolates belonging to the same sequence type (ST307). All KPN isolates harbored conjugative resistance plasmids, which has blaKPC-2 carbapenemase genes contained within the Tn4401 “a” isoform and other resistance genes. However, WGS showed only three isolates among four KPC-producing KPN were originated from a common origin. Conclusions: This report demonstrates the challenge that KPC-2-producing KPN with the conjugative resistance plasmid may spread not only in hospitals but also in community, and WGS can help to accurately characterize the outbreak.


April 21, 2020  |  

Complete genome sequence of Marinobacter sp. LQ44, a haloalkaliphilic phenol-degrading bacterium isolated from a deep-sea hydrothermal vent

Marinobacter sp. strain LQ44, an alkaliphile and moderate halophile from a deep-sea hydrothermal vent on the East Pacific Rise, is a novel phenol-degrading bacterium that is capable of utilizing phenol as sole carbon and energy sources. Here, we present the complete genome sequence of strain LQ44, which consists of 4,435,564?bp with a circular chromosome, 4164 protein-coding genes, 3 rRNA operons and 50 tRNAs. Genome analysis revealed that strain LQ44 may degrade phenol via meta-cleavage pathway. The LQ44 genome contains multiple genes involved in pH adaptation and osmotic adjustment. Genes related to hydrocarbon degradation, aerobic denitrification and potential industrial important enzymes were also identified from the genome. To our knowledge, this is the first report of a genome sequence of a haloalkaliphilic phenol-degrading bacterium, which will provide insights into the survival of this bacterium under salt-alkali conditions and the potential for biotechnological applications.


April 21, 2020  |  

A robust benchmark for germline structural variant detection

New technologies and analysis methods are enabling genomic structural variants (SVs) to be detected with ever-increasing accuracy, resolution, and comprehensiveness. Translating these methods to routine research and clinical practice requires robust benchmark sets. We developed the first benchmark set for identification of both false negative and false positive germline SVs, which complements recent efforts emphasizing increasingly comprehensive characterization of SVs. To create this benchmark for a broadly consented son in a Personal Genome Project trio with broadly available cells and DNA, the Genome in a Bottle (GIAB) Consortium integrated 19 sequence-resolved variant calling methods, both alignment- and de novo assembly-based, from short-, linked-, and long-read sequencing, as well as optical and electronic mapping. The final benchmark set contains 12745 isolated, sequence-resolved insertion and deletion calls =50 base pairs (bp) discovered by at least 2 technologies or 5 callsets, genotyped as heterozygous or homozygous variants by long reads. The Tier 1 benchmark regions, for which any extra calls are putative false positives, cover 2.66 Gbp and 9641 SVs supported by at least one diploid assembly. Support for SVs was assessed using svviz with short-, linked-, and long-read sequence data. In general, there was strong support from multiple technologies for the benchmark SVs, with 90 % of the Tier 1 SVs having support in reads from more than one technology. The Mendelian genotype error rate was 0.3 %, and genotype concordance with manual curation was >98.7 %. We demonstrate the utility of the benchmark set by showing it reliably identifies both false negatives and false positives in high-quality SV callsets from short-, linked-, and long-read sequencing and optical mapping.


April 21, 2020  |  

The Genome of the Zebra Mussel, Dreissena polymorpha: A Resource for Invasive Species Research

The zebra mussel, Dreissena polymorpha, continues to spread from its native range in Eurasia to Europe and North America, causing billions of dollars in damage and dramatically altering invaded aquatic ecosystems. Despite these impacts, there are few genomic resources for Dreissena or related bivalves, with nearly 450 million years of divergence between zebra mussels and its closest sequenced relative. Although the D. polymorpha genome is highly repetitive, we have used a combination of long-read sequencing and Hi-C-based scaffolding to generate the highest quality molluscan assembly to date. Through comparative analysis and transcriptomics experiments we have gained insights into processes that likely control the invasive success of zebra mussels, including shell formation, synthesis of byssal threads, and thermal tolerance. We identified multiple intact Steamer-Like Elements, a retrotransposon that has been linked to transmissible cancer in marine clams. We also found that D. polymorpha have an unusual 67 kb mitochondrial genome containing numerous tandem repeats, making it the largest observed in Eumetazoa. Together these findings create a rich resource for invasive species research and control efforts.


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