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April 21, 2020

ASA3P: An automatic and scalable pipeline for the assembly, annotation and higher level analysis of closely related bacterial isolates

Whole genome sequencing of bacteria has become daily routine in many fields. Advances in DNA sequencing technologies and continuously dropping costs have resulted in a tremendous increase in the amounts of available sequence data. However, comprehensive in-depth analysis of the resulting data remains an arduous and time consuming task. In order to keep pace with these promising but challenging developments and to transform raw data into valuable information, standardized analyses and scalable software tools are needed. Here, we introduce ASA3P, a fully automatic, locally executable and scalable assembly, annotation and analysis pipeline for bacterial genomes. The pipeline automatically executes necessary data processing steps, i.e. quality clipping and assembly of raw sequencing reads, scaffolding of contigs and annotation of the resulting genome sequences. Furthermore, ASA3P conducts comprehensive genome characterizations and analyses, e.g. taxonomic classification, detection of antibiotic resistance genes and identification of virulence factors. All results are presented via an HTML5 user interface providing aggregated information, interactive visualizations and access to intermediate results in standard bioinformatics file formats. We distribute ASA3P in two versions: a locally executable Docker container for small-to-medium-scale projects and an OpenStack based cloud computing version able to automatically create and manage self-scaling compute clusters. Thus, automatic and standardized analysis of hundreds of bacterial genomes becomes feasible within hours. The software and further information is available at: http://asap.computational.bio.

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April 21, 2020

Transcriptional initiation of a small RNA, not R-loop stability, dictates the frequency of pilin antigenic variation in Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the sole causative agent of gonorrhea, constitutively undergoes diversification of the Type IV pilus. Gene conversion occurs between one of the several donor silent copies located in distinct loci and the recipient pilE gene, encoding the major pilin subunit of the pilus. A guanine quadruplex (G4) DNA structure and a cis-acting sRNA (G4-sRNA) are located upstream of the pilE gene and both are required for pilin antigenic variation (Av). We show that the reduced sRNA transcription lowers pilin Av frequencies. Extended transcriptional elongation is not required for Av, since limiting the transcript to 32 nt allows for normal Av frequencies. Using chromatin immunoprecipitation (ChIP) assays, we show that cellular G4s are less abundant when sRNA transcription is lower. In addition, using ChIP, we demonstrate that the G4-sRNA forms a stable RNA:DNA hybrid (R-loop) with its template strand. However, modulating R-loop levels by controlling RNase HI expression does not alter G4 abundance quantified through ChIP. Since pilin Av frequencies were not altered when modulating R-loop levels by controlling RNase HI expression, we conclude that transcription of the sRNA is necessary, but stable R-loops are not required to promote pilin Av. © 2019 John Wiley & Sons Ltd.

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April 21, 2020

Chromosome-level hybrid de novo genome assemblies as an attainable option for non-model organisms

The emergence of third generation sequencing (3GS; long-reads) is making closer the goal of chromosome-size fragments in de novo genome assemblies. This allows the exploration of new and broader questions on genome evolution for a number of non-model organisms. However, long-read technologies result in higher sequencing error rates and therefore impose an elevated cost of sufficient coverage to achieve high enough quality. In this context, hybrid assemblies, combining short-reads and long-reads provide an alternative efficient and cost-effective approach to generate de novo, chromosome-level genome assemblies. The array of available software programs for hybrid genome assembly, sequence correction and manipulation is constantly being expanded and improved. This makes it difficult for non-experts to find efficient, fast and tractable computational solutions for genome assembly, especially in the case of non-model organisms lacking a reference genome or one from a closely related species. In this study, we review and test the most recent pipelines for hybrid assemblies, comparing the model organism Drosophila melanogaster to a non-model cactophilic Drosophila, D. mojavensis. We show that it is possible to achieve excellent contiguity on this non-model organism using the DBG2OLC pipeline.

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April 21, 2020

Acquired N-Linked Glycosylation Motifs in B-Cell Receptors of Primary Cutaneous B-Cell Lymphoma and the Normal B-Cell Repertoire.

Primary cutaneous follicle center lymphoma (PCFCL) is a rare mature B-cell lymphoma with an unknown etiology. PCFCL resembles follicular lymphoma (FL) by cytomorphologic and microarchitectural criteria. FL B cells are selected for N-linked glycosylation motifs in their B-cell receptors (BCRs) that are acquired during continuous somatic hypermutation. The stimulation of mannosylated BCR by lectins on the tumor microenvironment is therefore a candidate driver in FL pathogenesis. We investigated whether the same mechanism could play a role in PCFCL pathogenesis. Full-length functional variable, diversity, and joining gene sequences of 18 PCFCL and 8 primary cutaneous diffuse large B-cell lymphoma, leg-type were identified by unbiased Anchoring Reverse Transcription of Immunoglobulin Sequences and Amplification by Nested PCR and BCR reconstruction from RNA sequencing data. Low BCR variation demonstrated negligible ongoing somatic hypermutation in PCFCL and primary cutaneous diffuse large B-cell lymphoma, leg-type, and indicated that the PCFCL microarchitecture does not act as a functional germinal center. Similar to FL but in contrast to primary cutaneous diffuse large B-cell lymphoma, leg-type, BCR genes of 15 PCFCLs (83%) had acquired N-linked glycosylation motifs. These motifs were located at the BCR positions converted to N-linked glycosylation motifs in normal B-cell repertoires with low prevalence but mostly at different positions than those found in FL. The cutaneous localization of PCFCL might suggest a role for lectins from commensal skin bacteria in PCFCL lymphomagenesis.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.

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April 21, 2020

Plasmid-encoded tet(X) genes that confer high-level tigecycline resistance in Escherichia coli.

Tigecycline is one of the last-resort antibiotics to treat complicated infections caused by both multidrug-resistant Gram-negative and Gram-positive bacteria1. Tigecycline resistance has sporadically occurred in recent years, primarily due to chromosome-encoding mechanisms, such as overexpression of efflux pumps and ribosome protection2,3. Here, we report the emergence of the plasmid-mediated mobile tigecycline resistance mechanism Tet(X4) in Escherichia coli isolates from China, which is capable of degrading all tetracyclines, including tigecycline and the US FDA newly approved eravacycline. The tet(X4)-harbouring IncQ1 plasmid is highly transferable, and can be successfully mobilized and stabilized in recipient clinical and laboratory strains of Enterobacteriaceae bacteria. It is noteworthy that tet(X4)-positive E.?coli strains, including isolates co-harbouring mcr-1, have been widely detected in pigs, chickens, soil and dust samples in China. In vivo murine models demonstrated that the presence of Tet(X4) led to tigecycline treatment failure. Consequently, the emergence of plasmid-mediated Tet(X4) challenges the clinical efficacy of the entire family of tetracycline antibiotics. Importantly, our study raises concern that the plasmid-mediated tigecycline resistance may further spread into various ecological niches and into clinical high-risk pathogens. Collective efforts are in urgent need to preserve the potency of these essential antibiotics.

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April 21, 2020

Antibiotic susceptibility of plant-derived lactic acid bacteria conferring health benefits to human.

Lactic acid bacteria (LAB) confer health benefits to human when administered orally. We have recently isolated several species of LAB strains from plant sources, such as fruits, vegetables, flowers, and medicinal plants. Since antibiotics used to treat bacterial infection diseases induce the emergence of drug-resistant bacteria in intestinal microflora, it is important to evaluate the susceptibility of LAB strains to antibiotics to ensure the safety and security of processed foods. The aim of the present study is to determine the minimum inhibitory concentration (MIC) of antibiotics against several plant-derived LAB strains. When aminoglycoside antibiotics, such as streptomycin (SM), kanamycin (KM), and gentamicin (GM), were evaluated using LAB susceptibility test medium (LSM), the MIC was higher than when using Mueller-Hinton (MH) medium. Etest, which is an antibiotic susceptibility assay method consisting of a predefined gradient of antibiotic concentrations on a plastic strip, is used to determine the MIC of antibiotics world-wide. In the present study, we demonstrated that Etest was particularly valuable while testing LAB strains. We also show that the low susceptibility of the plant-derived LAB strains against each antibiotic tested is due to intrinsic resistance and not acquired resistance. This finding is based on the whole-genome sequence information reflecting the horizontal spread of the drug-resistance genes in the LAB strains.

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April 21, 2020

RNA sequencing: the teenage years.

Over the past decade, RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing of mRNAs. However, as next-generation sequencing technologies have developed, so too has RNA-seq. Now, RNA-seq methods are available for studying many different aspects of RNA biology, including single-cell gene expression, translation (the translatome) and RNA structure (the structurome). Exciting new applications are being explored, such as spatial transcriptomics (spatialomics). Together with new long-read and direct RNA-seq technologies and better computational tools for data analysis, innovations in RNA-seq are contributing to a fuller understanding of RNA biology, from questions such as when and where transcription occurs to the folding and intermolecular interactions that govern RNA function.

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April 21, 2020

Large Fragment Deletions Induced by Cas9 Cleavage While Not in BEs System in Rabbit

CRISPR-Cas9 and BEs system are poised to become the gene editing tool of choice in clinical contexts, however large fragment deletion was found in Cas9-mediated mutation cells without animal level validation. By analyzing 16 gene-edited rabbit lines (including 112 rabbits) generated using SpCas9, BEs, xCas9 and xCas9-BEs with long-range PCR genotyping and long-read sequencing by PacBio platform, we show that extending thousands of bases fragment deletions in single-guide RNA/Cas9 and xCas9 system mutation rabbit, but few large deletions were found in BEs-induced mutation rabbits. We firstly validated that no large fragment deletion induced by BEs system at animal level, suggesting that BE systems can be beneficial tools for the further development of highly accurate and secure gene therapy for the clinical treatment of human genetic disorders

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April 21, 2020

Comparison of mitochondrial DNA variants detection using short- and long-read sequencing.

The recent advent of long-read sequencing technologies is expected to provide reasonable answers to genetic challenges unresolvable by short-read sequencing, primarily the inability to accurately study structural variations, copy number variations, and homologous repeats in complex parts of the genome. However, long-read sequencing comes along with higher rates of random short deletions and insertions, and single nucleotide errors. The relatively higher sequencing accuracy of short-read sequencing has kept it as the first choice of screening for single nucleotide variants and short deletions and insertions. Albeit, short-read sequencing still suffers from systematic errors that tend to occur at specific positions where a high depth of reads is not always capable to correct for these errors. In this study, we compared the genotyping of mitochondrial DNA variants in three samples using PacBio’s Sequel (Pacific Biosciences Inc., Menlo Park, CA, USA) long-read sequencing and illumina’s HiSeqX10 (illumine Inc., San Diego, CA, USA) short-read sequencing data. We concluded that, despite the differences in the type and frequency of errors in the long-reads sequencing, its accuracy is still comparable to that of short-reads for genotyping short nuclear variants; due to the randomness of errors in long reads, a lower coverage, around 37 reads, can be sufficient to correct for these random errors.

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April 21, 2020

The Chinese chestnut genome: a reference for species restoration

Forest tree species are increasingly subject to severe mortalities from exotic pests, diseases, and invasive organisms, accelerated by climate change. Forest health issues are threatening multiple species and ecosystem sustainability globally. While sources of resistance may be available in related species, or among surviving trees, introgression of resistance genes into threatened tree species in reasonable time frames requires genome-wide breeding tools. Asian species of chestnut (Castanea spp.) are being employed as donors of disease resistance genes to restore native chestnut species in North America and Europe. To aid in the restoration of threatened chestnut species, we present the assembly of a reference genome with chromosome-scale sequences for Chinese chestnut (C. mollissima), the disease-resistance donor for American chestnut restoration. We also demonstrate the value of the genome as a platform for research and species restoration, including new insights into the evolution of blight resistance in Asian chestnut species, the locations in the genome of ecologically important signatures of selection differentiating American chestnut from Chinese chestnut, the identification of candidate genes for disease resistance, and preliminary comparisons of genome organization with related species.

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April 21, 2020

Benchmarking Transposable Element Annotation Methods for Creation of a Streamlined, Comprehensive Pipeline

Sequencing technology and assembly algorithms have matured to the point that high-quality de novo assembly is possible for large, repetitive genomes. Current assemblies traverse transposable elements (TEs) and allow for annotation of TEs. There are numerous methods for each class of elements with unknown relative performance metrics. We benchmarked existing programs based on a curated library of rice TEs. Using the most robust programs, we created a comprehensive pipeline called Extensive de-novo TE Annotator (EDTA) that produces a condensed TE library for annotations of structurally intact and fragmented elements. EDTA is open-source and freely available: https://github.com/oushujun/EDTA.List of abbreviationsTETransposable ElementsLTRLong Terminal RepeatLINELong Interspersed Nuclear ElementSINEShort Interspersed Nuclear ElementMITEMiniature Inverted Transposable ElementTIRTerminal Inverted RepeatTSDTarget Site DuplicationTPTrue PositivesFPFalse PositivesTNTrue NegativeFNFalse NegativesGRFGeneric Repeat FinderEDTAExtensive de-novo TE Annotator

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April 21, 2020

Insights into the bacterial species and communities of a full-scale anaerobic/anoxic/oxic wastewater treatment plant by using third-generation sequencing.

For the first time, full-length 16S rRNA sequencing method was applied to disclose the bacterial species and communities of a full-scale wastewater treatment plant using an anaerobic/anoxic/oxic (A/A/O) process in Wuhan, China. The compositions of the bacteria at phylum and class levels in the activated sludge were similar to which revealed by Illumina Miseq sequencing. At genus and species levels, third-generation sequencing showed great merits and accuracy. Typical functional taxa classified to ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), denitrifying bacteria (DB), anaerobic ammonium oxidation bacteria (ANAMMOXB) and polyphosphate-accumulating organisms (PAOs) were presented, which were Nitrosomonas (1.11%), Nitrospira (3.56%), Pseudomonas (3.88%), Planctomycetes (13.80%), Comamonadaceae (1.83%), respectively. Pseudomonas (3.88%) and Nitrospira (3.56%) were the most predominating two genera, mainly containing Pseudomonas extremaustralis (1.69%), Nitrospira defluvii (3.13%), respectively. Bacteria regarding to nitrogen and phosphorus removal at species level were put forward. The predicted functions proved that the A/A/O process was efficient regarding nitrogen and organics removal. Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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April 21, 2020

Genomic analysis of Marinobacter sp. NP-4 and NP-6 isolated from the deep-sea oceanic crust on the western flank of the Mid-Atlantic Ridge

Two Marinobacter sp. NP-4 and NP-6 were isolated from a deep oceanic basaltic crust at North Pond, located at the western flank of the Mid-Atlantic Ridge. These two strains are capable of using multiple carbon sources such as acetate, succinate, glucose and sucrose while take oxygen as a primary electron acceptor. The strain NP-4 is also able to grow anaerobically under 20?MPa, with nitrate as the electron acceptor, thus represents a piezotolerant. To explore the metabolic potentials of Marinobacter sp. NP-4 and NP-6, the complete genome of NP-4 and close-to-complete genome of NP-6 were sequenced. The genome of NP-4 contains one chromosome and two plasmids with the size of 4.6?Mb in total, and with average GC content of 57.0%. The genome of NP-6 is 4.5?Mb and consists of 6 scaffolds, with an average GC content of 57.1%. Complete glycolysis, citrate cycle and aromatics compounds degradation pathways are identified in genomes of these two strains, suggesting that they possess a heterotrophic life style. Additionally, one plasmid of NP-4 contains genes for alkane degradation, phosphonate ABC transporter and cation efflux system, enabling NP-4 extra surviving abilities. In total, genomic information of these two strains provide insights into the physiological features and adaptation strategies of Marinobacter spp. in the deep oceanic crust biosphere.

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April 21, 2020

Variant Phasing and Haplotypic Expression from Single-molecule Long-read Sequencing in Maize

Haplotype phasing of genetic variants is important for interpretation of the maize genome, population genetic analysis, and functional genomic analysis of allelic activity. Accordingly, accurate methods for phasing full-length isoforms are essential for functional genomics study. In this study, we performed an isoform-level phasing study in maize, using two inbred lines and their reciprocal crosses, based on single-molecule full-length cDNA sequencing. To phase and analyze full-length transcripts between hybrids and parents, we developed a tool called IsoPhase. Using this tool, we validated the majority of SNPs called against matching short read data and identified cases of allele-specific, gene-level, and isoform-level expression. Our results revealed that maize parental and hybrid lines exhibit different splicing activities. After phasing 6,847 genes in two reciprocal hybrids using embryo, endosperm and root tissues, we annotated the SNPs and identified large-effect genes. In addition, based on single-molecule sequencing, we identified parent-of-origin isoforms in maize hybrids, different novel isoforms between maize parent and hybrid lines, and imprinted genes from different tissues. Finally, we characterized variation in cis- and trans-regulatory effects. Our study provides measures of haplotypic expression that could increase power and accuracy in studies of allelic expression.

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April 21, 2020

Genome sequence resource for Ilyonectria mors-panacis, causing rusty root rot of Panax notoginseng.

Ilyonectria mors-panacis is a serious disease hampering the production of Panax notoginseng, an important Chinese medicinal herb, widely used for its anti-inflammatory, anti-fatigue, hepato-protective, and coronary heart disease prevention effects. Here, we report the first Illumina-Pacbio hybrid sequenced draft genome assembly of I. mors-panacis strain G3B and its annotation. The availability of this genome sequence not only represents an important tool toward understanding the genetics behind the infection mechanism of I. mors-panacis strain G3B but also will help illuminate the complexities of the taxonomy of this species.

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