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Auto Tag: Model organism

September 22, 2019  |  

Effects of antibiotic on microflora in ileum and cecum for broilers by 16S rRNA sequence analysis.

An experiment was conducted to analyze and compare the microbial composition, abundance, dynamic distribution, and functions without and with antibiotic fed to broilers. A 16S rRNA-sequencing approach was used to evaluate the bacterial composition of the gut of male broilers under different groups. A total of 240 1-day old AA male broilers were randomly assigned to two groups, with 120 broilers per group. The treatment group was administered an antibiotic with their feed, while the control group was not administered antibiotic (control group). A total of 10 replicates were assessed per treatment. The control group was fed a basal diet containing corn, soybean meal, and cottonseed meal and met the nutritional requirement. The antibiotic group was fed 100 mg/kg aureomycin (based on the basal diet). The trial lasted 42 days. Operational taxonomic unit partition and classification, alpha diversity, taxonomic composition, beta diversity, and microflora comparative analyses along with key species screening were performed for all of the treatment groups. Our data indicate that aureomycin treatment in broilers is directly correlated with variations of the gut content of specific bacterial taxa, and herein provide insights into the impact of antibiotic on microbial communities in cecum and ileum of broiler chickens.© 2018 Japanese Society of Animal Science.

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September 22, 2019  |  

Long-read based assembly and annotation of a Drosophila simulans genome

Long-read sequencing technologies enable high-quality, contiguous genome assemblies. Here we used SMRT sequencing to assemble the genome of a Drosophila simulans strain originating from Madagascar, the ancestral range of the species. We generated 8 Gb of raw data (~50x coverage) with a mean read length of 6,410 bp, a NR50 of 9,125 bp and the longest subread at 49 kb. We benchmarked six different assemblers and merged the best two assemblies from Canu and Falcon. Our final assembly was 127.41 Mb with a N50 of 5.38 Mb and 305 contigs. We anchored more than 4 Mb of novel sequence to the major chromosome arms, and significantly improved the assembly of peri-centromeric and telomeric regions. Finally, we performed full-length transcript sequencing and used this data in conjunction with short-read RNAseq data to annotate 13,422 genes in the genome, improving the annotation in regions with complex, nested gene structures.

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September 22, 2019  |  

Transcriptomic study of Herpes simplex virus type-1 using full-length sequencing techniques

Herpes simplex virus type-1 (HSV-1) is a human pathogenic member of the Alphaherpesvirinae subfamily of herpesviruses. The HSV-1 genome is a large double-stranded DNA specifying about 85 protein coding genes. The latest surveys have demonstrated that the HSV-1 transcriptome is much more complex than it had been thought before. Here, we provide a long-read sequencing dataset, which was generated by using the RSII and Sequel systems from Pacific Biosciences (PacBio), as well as MinION sequencing system from Oxford Nanopore Technologies (ONT). This dataset contains 39,096 reads of inserts (ROIs) mapped to the HSV-1 genome (X14112) in RSII sequencing, while Sequel sequencing yielded 77,851 ROIs. The MinION cDNA sequencing altogether resulted in 158,653 reads, while the direct RNA-seq produced 16,516 reads. This dataset can be utilized for the identification of novel HSV RNAs and transcripts isoforms, as well as for the comparison of the quality and length of the sequencing reads derived from the currently available long- read sequencing platforms. The various library preparation approaches can also be compared with each other.

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September 22, 2019  |  

Anthropogenic N deposition alters the composition of expressed class II fungal peroxidases.

Here, we present evidence that ca. 20 years of experimental N deposition altered the composition of lignin-decaying class II peroxidases expressed by forest floor fungi, a response which has occurred concurrently with reductions in plant litter decomposition and a rapid accumulation of soil organic matter. This finding suggests that anthropogenic N deposition has induced changes in the biological mediation of lignin decay, the rate limiting step in plant litter decomposition. Thus, an altered composition of transcripts for a critical gene that is associated with terrestrial C cycling may explain the increased soil C storage under long-term increases in anthropogenic N deposition.IMPORTANCE Fungal class II peroxidases are enzymes that mediate the rate-limiting step in the decomposition of plant material, which involves the oxidation of lignin and other polyphenols. In field experiments, anthropogenic N deposition has increased soil C storage in forests, a result which could potentially arise from anthropogenic N-induced changes in the composition of class II peroxidases expressed by the fungal community. In this study, we have gained unique insight into how anthropogenic N deposition, a widespread agent of global change, affects the expression of a functional gene encoding an enzyme that plays a critical role in a biologically mediated ecosystem process. Copyright © 2018 American Society for Microbiology.

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September 22, 2019  |  

Bypassing the Restriction System To Improve Transformation of Staphylococcus epidermidis.

Staphylococcus epidermidis is the leading cause of infections on indwelling medical devices worldwide. Intrinsic antibiotic resistance and vigorous biofilm production have rendered these infections difficult to treat and, in some cases, require the removal of the offending medical prosthesis. With the exception of two widely passaged isolates, RP62A and 1457, the pathogenesis of infections caused by clinical S. epidermidis strains is poorly understood due to the strong genetic barrier that precludes the efficient transformation of foreign DNA into clinical isolates. The difficulty in transforming clinical S. epidermidis isolates is primarily due to the type I and IV restriction-modification systems, which act as genetic barriers. Here, we show that efficient plasmid transformation of clinical S. epidermidis isolates from clonal complexes 2, 10, and 89 can be realized by employing a plasmid artificial modification (PAM) in Escherichia coli DC10B containing a ?dcm mutation. This transformative technique should facilitate our ability to genetically modify clinical isolates of S. epidermidis and hence improve our understanding of their pathogenesis in human infections.IMPORTANCEStaphylococcus epidermidis is a source of considerable morbidity worldwide. The underlying mechanisms contributing to the commensal and pathogenic lifestyles of S. epidermidis are poorly understood. Genetic manipulations of clinically relevant strains of S. epidermidis are largely prohibited due to the presence of a strong restriction barrier. With the introductions of the tools presented here, genetic manipulation of clinically relevant S. epidermidis isolates has now become possible, thus improving our understanding of S. epidermidis as a pathogen. Copyright © 2017 American Society for Microbiology.

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September 22, 2019  |  

Single-molecule long-read transcriptome dataset of halophyte Halogeton glomeratus.

Soil salinization has become a major challenge for sustainable development of global agriculture. As a result, cultivation of salt-tolerant crop varieties has become a focus of plant breeding. However, development of effective breeding strategies would be significantly enhanced by improving our understanding of salt tolerance mechanisms in plants and identifying genes required for adaptation.

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September 22, 2019  |  

Comparative Annotation Toolkit (CAT)-simultaneous clade and personal genome annotation.

The recent introductions of low-cost, long-read, and read-cloud sequencing technologies coupled with intense efforts to develop efficient algorithms have made affordable, high-quality de novo sequence assembly a realistic proposition. The result is an explosion of new, ultracontiguous genome assemblies. To compare these genomes, we need robust methods for genome annotation. We describe the fully open source Comparative Annotation Toolkit (CAT), which provides a flexible way to simultaneously annotate entire clades and identify orthology relationships. We show that CAT can be used to improve annotations on the rat genome, annotate the great apes, annotate a diverse set of mammals, and annotate personal, diploid human genomes. We demonstrate the resulting discovery of novel genes, isoforms, and structural variants-even in genomes as well studied as rat and the great apes-and how these annotations improve cross-species RNA expression experiments.© 2018 Fiddes et al.; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019  |  

Normalized long read RNA sequencing in chicken reveals transcriptome complexity similar to human.

Despite the significance of chicken as a model organism, our understanding of the chicken transcriptome is limited compared to human. This issue is common to all non-human vertebrate annotations due to the difficulty in transcript identification from short read RNAseq data. While previous studies have used single molecule long read sequencing for transcript discovery, they did not perform RNA normalization and 5′-cap selection which may have resulted in lower transcriptome coverage and truncated transcript sequences.We sequenced normalised chicken brain and embryo RNA libraries with Pacific Bioscience Iso-Seq. 5′ cap selection was performed on the embryo library to provide methodological comparison. From these Iso-Seq sequencing projects, we have identified 60 k transcripts and 29 k genes within the chicken transcriptome. Of these, more than 20 k are novel lncRNA transcripts with ~3 k classified as sense exonic overlapping lncRNA, which is a class that is underrepresented in many vertebrate annotations. The relative proportion of alternative transcription events revealed striking similarities between the chicken and human transcriptomes while also providing explanations for previously observed genomic differences.Our results indicate that the chicken transcriptome is similar in complexity compared to human, and provide insights into other vertebrate biology. Our methodology demonstrates the potential of Iso-Seq sequencing to rapidly expand our knowledge of transcriptomics.

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September 22, 2019  |  

Avian transcriptomics: opportunities and challenges

Recent developments in next-generation sequencing technologies have greatly facilitated the study of whole transcriptomes in model and non-model species. Studying the transcriptome and how it changes across a variety of biological conditions has had major implications for our understanding of how the genome is regulated in different contexts, and how to interpret adaptations and the phenotype of an organism. The aim of this review is to highlight the potential of these new technologies for the study of avian transcriptomics, and to summarise how transcriptomics has been applied in ornithology. A total of 81 peer-reviewed scientific articles that used transcriptomics to answer questions within a broad range of study areas in birds are used as examples throughout the review. We further provide a quick guide to highlight the most important points which need to be take into account when planning a transcriptomic study in birds, and discuss how researchers with little background in molecular biology can avoid potential pitfalls. Suggestions for further reading are supplied throughout. We also discuss possible future developments in the technology platforms used for ribonucleic acid sequencing. By summarising how these novel technologies can be used to answer questions that have long been asked by ornithologists, we hope to bridge the gap between traditional ornithology and genomics, and to stimulate more interdisciplinary research.

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September 22, 2019  |  

Multi-platform sequencing approach reveals a novel transcriptome profile in pseudorabies virus.

Third-generation sequencing is an emerging technology that is capable of solving several problems that earlier approaches were not able to, including the identification of transcripts isoforms and overlapping transcripts. In this study, we used long-read sequencing for the analysis of pseudorabies virus (PRV) transcriptome, including Oxford Nanopore Technologies MinION, PacBio RS-II, and Illumina HiScanSQ platforms. We also used data from our previous short-read and long-read sequencing studies for the comparison of the results and in order to confirm the obtained data. Our investigations identified 19 formerly unknown putative protein-coding genes, all of which are 5′ truncated forms of earlier annotated longer PRV genes. Additionally, we detected 19 non-coding RNAs, including 5′ and 3′ truncated transcripts without in-frame ORFs, antisense RNAs, as well as RNA molecules encoded by those parts of the viral genome where no transcription had been detected before. This study has also led to the identification of three complex transcripts and 50 distinct length isoforms, including transcription start and end variants. We also detected 121 novel transcript overlaps, and two transcripts that overlap the replication origins of PRV. Furthermore,in silicoanalysis revealed 145 upstream ORFs, many of which are located on the longer 5′ isoforms of the transcripts.

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September 22, 2019  |  

Meeting report: 31st International Mammalian Genome Conference, Mammalian Genetics and Genomics: From Molecular Mechanisms to Translational Applications.

High on the Heidelberg hills, inside the Advanced Training Centre of the European Molecular Biology Laboratory (EMBL) campus with its unique double-helix staircase, scientists gathered for the EMBL conference “Mammalian Genetics and Genomics: From Molecular Mechanisms to Translational Applications,” organized in cooperation with the International Mammalian Genome Society (IMGS) and the Mouse Molecular Genetics (MMG) group. The conference attracted 205 participants from 30 countries, representing 6 of the 7 continents-all except Antarctica. It was a richly diverse group of geneticists, clinicians, and bioinformaticians, with presentations by established and junior investigators, including many trainees. From the 24th-27th of October 2017, they shared exciting advances in mammalian genetics and genomics research, from the introduction of cutting-edge technologies to descriptions of translational studies involving highly relevant models of human disease.

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September 22, 2019  |  

Young genes have distinct gene structure, epigenetic profiles, and transcriptional regulation.

Species-specific, new, or “orphan” genes account for 10%-30% of eukaryotic genomes. Although initially considered to have limited function, an increasing number of orphan genes have been shown to provide important phenotypic innovation. How new genes acquire regulatory sequences for proper temporal and spatial expression is unknown. Orphan gene regulation may rely in part on origination in open chromatin adjacent to preexisting promoters, although this has not yet been assessed by genome-wide analysis of chromatin states. Here, we combine taxon-rich nematode phylogenies with Iso-Seq, RNA-seq, ChIP-seq, and ATAC-seq to identify the gene structure and epigenetic signature of orphan genes in the satellite model nematode Pristionchus pacificus Consistent with previous findings, we find young genes are shorter, contain fewer exons, and are on average less strongly expressed than older genes. However, the subset of orphan genes that are expressed exhibit distinct chromatin states from similarly expressed conserved genes. Orphan gene transcription is determined by a lack of repressive histone modifications, confirming long-held hypotheses that open chromatin is important for new gene formation. Yet orphan gene start sites more closely resemble enhancers defined by H3K4me1, H3K27ac, and ATAC-seq peaks, in contrast to conserved genes that exhibit traditional promoters defined by H3K4me3 and H3K27ac. Although the majority of orphan genes are located on chromosome arms that contain high recombination rates and repressive histone marks, strongly expressed orphan genes are more randomly distributed. Our results support a model of new gene origination by rare integration into open chromatin near enhancers.© 2018 Werner et al.; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019  |  

The state of play in higher eukaryote gene annotation.

A genome sequence is worthless if it cannot be deciphered; therefore, efforts to describe – or ‘annotate’ – genes began as soon as DNA sequences became available. Whereas early work focused on individual protein-coding genes, the modern genomic ocean is a complex maelstrom of alternative splicing, non-coding transcription and pseudogenes. Scientists – from clinicians to evolutionary biologists – need to navigate these waters, and this has led to the design of high-throughput, computationally driven annotation projects. The catalogues that are being produced are key resources for genome exploration, especially as they become integrated with expression, epigenomic and variation data sets. Their creation, however, remains challenging.

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September 22, 2019  |  

SuperTranscripts: a data driven reference for analysis and visualisation of transcriptomes.

Numerous methods have been developed to analyse RNA sequencing (RNA-seq) data, but most rely on the availability of a reference genome, making them unsuitable for non-model organisms. Here we present superTranscripts, a substitute for a reference genome, where each gene with multiple transcripts is represented by a single sequence. The Lace software is provided to construct superTranscripts from any set of transcripts, including de novo assemblies. We demonstrate how superTranscripts enable visualisation, variant detection and differential isoform detection in non-model organisms. We further use Lace to combine reference and assembled transcriptomes for chicken and recover hundreds of gaps in the reference genome.

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September 22, 2019  |  

Genome re-annotation of the wild strawberry Fragaria vesca using extensive Illumina-and SMRT-based RNA-seq datasets

The genome of the wild diploid strawberry species Fragaria vesca, an ideal model system of cultivated strawberry (Fragaria × ananassa, octoploid) and other Rosaceae family crops, was first published in 2011 and followed by a new assembly (Fvb). However, the annotation for Fvb mainly relied on ab initio predictions and included only predicted coding sequences, therefore an improved annotation is highly desirable. Here, a new annotation version named v2.0.a2 was created for the Fvb genome by a pipeline utilizing one PacBio library, 90 Illumina RNA-seq libraries, and 9 small RNA-seq libraries. Altogether, 18,641 genes (55.6% out of 33,538 genes) were augmented with information on the 5′ and/or 3′ UTRs, 13,168 (39.3%) protein-coding genes were modified or newly identified, and 7,370 genes were found to possess alternative isoforms. In addition, 1,938 long non-coding RNAs, 171 miRNAs, and 51,714 small RNA clusters were integrated into the annotation. This new annotation of F. vesca is substantially improved in both accuracy and integrity of gene predictions, beneficial to the gene functional studies in strawberry and to the comparative genomic analysis of other horticultural crops in Rosaceae family.

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