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Auto Tag: Drosophila melanogaster

September 22, 2019  |  

PLEK: a tool for predicting long non-coding RNAs and messenger RNAs based on an improved k-mer scheme.

High-throughput transcriptome sequencing (RNA-seq) technology promises to discover novel protein-coding and non-coding transcripts, particularly the identification of long non-coding RNAs (lncRNAs) from de novo sequencing data. This requires tools that are not restricted by prior gene annotations, genomic sequences and high-quality sequencing.We present an alignment-free tool called PLEK (predictor of long non-coding RNAs and messenger RNAs based on an improved k-mer scheme), which uses a computational pipeline based on an improved k-mer scheme and a support vector machine (SVM) algorithm to distinguish lncRNAs from messenger RNAs (mRNAs), in the absence of genomic sequences or annotations. The performance of PLEK was evaluated on well-annotated mRNA and lncRNA transcripts. 10-fold cross-validation tests on human RefSeq mRNAs and GENCODE lncRNAs indicated that our tool could achieve accuracy of up to 95.6%. We demonstrated the utility of PLEK on transcripts from other vertebrates using the model built from human datasets. PLEK attained >90% accuracy on most of these datasets. PLEK also performed well using a simulated dataset and two real de novo assembled transcriptome datasets (sequenced by PacBio and 454 platforms) with relatively high indel sequencing errors. In addition, PLEK is approximately eightfold faster than a newly developed alignment-free tool, named Coding-Non-Coding Index (CNCI), and 244 times faster than the most popular alignment-based tool, Coding Potential Calculator (CPC), in a single-threading running manner.PLEK is an efficient alignment-free computational tool to distinguish lncRNAs from mRNAs in RNA-seq transcriptomes of species lacking reference genomes. PLEK is especially suitable for PacBio or 454 sequencing data and large-scale transcriptome data. Its open-source software can be freely downloaded from https://sourceforge.net/projects/plek/files/.

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September 22, 2019  |  

Evaluation of tools for long read RNA-seq splice-aware alignment.

High-throughput sequencing has transformed the study of gene expression levels through RNA-seq, a technique that is now routinely used by various fields, such as genetic research or diagnostics. The advent of third generation sequencing technologies providing significantly longer reads opens up new possibilities. However, the high error rates common to these technologies set new bioinformatics challenges for the gapped alignment of reads to their genomic origin. In this study, we have explored how currently available RNA-seq splice-aware alignment tools cope with increased read lengths and error rates. All tested tools were initially developed for short NGS reads, but some have claimed support for long Pacific Biosciences (PacBio) or even Oxford Nanopore Technologies (ONT) MinION reads.The tools were tested on synthetic and real datasets from two technologies (PacBio and ONT MinION). Alignment quality and resource usage were compared across different aligners. The effect of error correction of long reads was explored, both using self-correction and correction with an external short reads dataset. A tool was developed for evaluating RNA-seq alignment results. This tool can be used to compare the alignment of simulated reads to their genomic origin, or to compare the alignment of real reads to a set of annotated transcripts. Our tests show that while some RNA-seq aligners were unable to cope with long error-prone reads, others produced overall good results. We further show that alignment accuracy can be improved using error-corrected reads.https://github.com/kkrizanovic/RNAseqEval, https://figshare.com/projects/RNAseq_benchmark/24391.mile.sikic@fer.hr.Supplementary data are available at Bioinformatics online.© The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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September 22, 2019  |  

Laboratory colonization stabilizes the naturally dynamic microbiome composition of field collected Dermacentor andersoni ticks.

Nearly a quarter of emerging infectious diseases identified in the last century are arthropod-borne. Although ticks and insects can carry pathogenic microorganisms, non-pathogenic microbes make up the majority of their microbial communities. The majority of tick microbiome research has had a focus on discovery and description; very few studies have analyzed the ecological context and functional responses of the bacterial microbiome of ticks. The goal of this analysis was to characterize the stability of the bacterial microbiome of Dermacentor andersoni ticks between generations and two populations within a species.The bacterial microbiome of D. andersoni midguts and salivary glands was analyzed from populations collected at two different ecologically distinct sites by comparing field (F1) and lab-reared populations (F1-F3) over three generations. The microbiome composition of pooled and individual samples was analyzed by sequencing nearly full-length 16S rRNA gene amplicons using a Pacific Biosciences CCS platform that allows identification of bacteria to the species level.In this study, we found that the D. andersoni microbiome was distinct in different geographic populations and was tissue specific, differing between the midgut and the salivary gland, over multiple generations. Additionally, our study showed that the microbiomes of laboratory-reared populations were not necessarily representative of their respective field populations. Furthermore, we demonstrated that the microbiome of a few individual ticks does not represent the microbiome composition at the population level.We demonstrated that the bacterial microbiome of D. andersoni was complex over three generations and specific to tick tissue (midgut vs. salivary glands) as well as geographic location (Burns, Oregon vs. Lake Como, Montana vs. laboratory setting). These results provide evidence that habitat of the tick population is a vital component of the complexity of the bacterial microbiome of ticks, and that the microbiome of lab colonies may not allow for comparative analyses with field populations. A broader understanding of microbiome variation will be required if we are to employ manipulation of the microbiome as a method for interfering with acquisition and transmission of tick-borne pathogens.

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September 22, 2019  |  

Comparative genome and transcriptome analysis reveals distinctive surface characteristics and unique physiological potentials of Pseudomonas aeruginosa ATCC 27853.

Pseudomonas aeruginosa ATCC 27853 was isolated from a hospital blood specimen in 1971 and has been widely used as a model strain to survey antibiotics susceptibilities, biofilm development, and metabolic activities of Pseudomonas spp.. Although four draft genomes of P. aeruginosa ATCC 27853 have been sequenced, the complete genome of this strain is still lacking, hindering a comprehensive understanding of its physiology and functional genome.Here we sequenced and assembled the complete genome of P. aeruginosa ATCC 27853 using the Pacific Biosciences SMRT (PacBio) technology and Illumina sequencing platform. We found that accessory genes of ATCC 27853 including prophages and genomic islands (GIs) mainly contribute to the difference between P. aeruginosa ATCC 27853 and other P. aeruginosa strains. Seven prophages were identified within the genome of P. aeruginosa ATCC 27853. Of the predicted 25 GIs, three contain genes that encode monoxoygenases, dioxygenases and hydrolases that could be involved in the metabolism of aromatic compounds. Surveying virulence-related genes revealed that a series of genes that encode the B-band O-antigen of LPS are lacking in ATCC 27853. Distinctive SNPs in genes of cellular adhesion proteins such as type IV pili and flagella biosynthesis were also observed in this strain. Colony morphology analysis confirmed an enhanced biofilm formation capability of ATCC 27853 on solid agar surface compared to Pseudomonas aeruginosa PAO1. We then performed transcriptome analysis of ATCC 27853 and PAO1 using RNA-seq and compared the expression of orthologous genes to understand the functional genome and the genomic details underlying the distinctive colony morphogenesis. These analyses revealed an increased expression of genes involved in cellular adhesion and biofilm maturation such as type IV pili, exopolysaccharide and electron transport chain components in ATCC 27853 compared with PAO1. In addition, distinctive expression profiles of the virulence genes lecA, lasB, quorum sensing regulators LasI/R, and the type I, III and VI secretion systems were observed in the two strains.The complete genome sequence of P. aeruginosa ATCC 27853 reveals the comprehensive genetic background of the strain, and provides genetic basis for several interesting findings about the functions of surface associated proteins, prophages, and genomic islands. Comparative transcriptome analysis of P. aeruginosa ATCC 27853 and PAO1 revealed several classes of differentially expressed genes in the two strains, underlying the genetic and molecular details of several known and yet to be explored morphological and physiological potentials of P. aeruginosa ATCC 27853.

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September 22, 2019  |  

GMAP and GSNAP for genomic sequence alignment: enhancements to speed, accuracy, and functionality.

The programs GMAP and GSNAP, for aligning RNA-Seq and DNA-Seq datasets to genomes, have evolved along with advances in biological methodology to handle longer reads, larger volumes of data, and new types of biological assays. The genomic representation has been improved to include linear genomes that can compare sequences using single-instruction multiple-data (SIMD) instructions, compressed genomic hash tables with fast access using SIMD instructions, handling of large genomes with more than four billion bp, and enhanced suffix arrays (ESAs) with novel data structures for fast access. Improvements to the algorithms have included a greedy match-and-extend algorithm using suffix arrays, segment chaining using genomic hash tables, diagonalization using segmental hash tables, and nucleotide-level dynamic programming procedures that use SIMD instructions and eliminate the need for F-loop calculations. Enhancements to the functionality of the programs include standardization of indel positions, handling of ambiguous splicing, clipping and merging of overlapping paired-end reads, and alignments to circular chromosomes and alternate scaffolds. The programs have been adapted for use in pipelines by integrating their usage into R/Bioconductor packages such as gmapR and HTSeqGenie, and these pipelines have facilitated the discovery of numerous biological phenomena.

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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  |  

Complete genome sequence of Enterococcus durans Oregon-R-modENCODE strain BDGP3, a lactic acid bacterium found in the Drosophila melanogaster gut

Enterococcus durans Oregon-R-modENCODE strain BDGP3 was isolated from the Drosophila melanogaster gut for functional host-microbe interaction studies. The complete genome is composed of a single circular genome of 2,983,334 bp, with a G+C content of 38%, and a single plasmid of 5,594 bp. Copyright © 2017 Wan et al.

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September 22, 2019  |  

Quantitative profiling of Drosophila melanogaster Dscam1 isoforms reveals no changes in splicing after bacterial exposure.

The hypervariable Dscam1 (Down syndrome cell adhesion molecule 1) gene can produce thousands of different ectodomain isoforms via mutually exclusive alternative splicing. Dscam1 appears to be involved in the immune response of some insects and crustaceans. It has been proposed that the diverse isoforms may be involved in the recognition of, or the defence against, diverse parasite epitopes, although evidence to support this is sparse. A prediction that can be generated from this hypothesis is that the gene expression of specific exons and/or isoforms is influenced by exposure to an immune elicitor. To test this hypothesis, we for the first time, use a long read RNA sequencing method to directly investigate the Dscam1 splicing pattern after exposing adult Drosophila melanogaster and a S2 cell line to live Escherichia coli. After bacterial exposure both models showed increased expression of immune-related genes, indicating that the immune system had been activated. However there were no changes in total Dscam1 mRNA expression. RNA sequencing further showed that there were no significant changes in individual exon expression and no changes in isoform splicing patterns in response to bacterial exposure. Therefore our studies do not support a change of D. melanogaster Dscam1 isoform diversity in response to live E. coli. Nevertheless, in future this approach could be used to identify potentially immune-related Dscam1 splicing regulation in other host species or in response to other pathogens.

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September 22, 2019  |  

Long-read sequencing of chicken transcripts and identification of new transcript isoforms.

The chicken has long served as an important model organism in many fields, and continues to aid our understanding of animal development. Functional genomics studies aimed at probing the mechanisms that regulate development require high-quality genomes and transcript annotations. The quality of these resources has improved dramatically over the last several years, but many isoforms and genes have yet to be identified. We hope to contribute to the process of improving these resources with the data presented here: a set of long cDNA sequencing reads, and a curated set of new genes and transcript isoforms not currently represented in the most up-to-date genome annotation currently available to the community of researchers who rely on the chicken genome.

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September 22, 2019  |  

Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing.

Neurexins are evolutionarily conserved presynaptic cell-adhesion molecules that are essential for normal synapse formation and synaptic transmission. Indirect evidence has indicated that extensive alternative splicing of neurexin mRNAs may produce hundreds if not thousands of neurexin isoforms, but no direct evidence for such diversity has been available. Here we use unbiased long-read sequencing of full-length neurexin (Nrxn)1a, Nrxn1ß, Nrxn2ß, Nrxn3a, and Nrxn3ß mRNAs to systematically assess how many sites of alternative splicing are used in neurexins with a significant frequency, and whether alternative splicing events at these sites are independent of each other. In sequencing more than 25,000 full-length mRNAs, we identified a novel, abundantly used alternatively spliced exon of Nrxn1a and Nrxn3a (referred to as alternatively spliced sequence 6) that encodes a 9-residue insertion in the flexible hinge region between the fifth LNS (laminin-a, neurexin, sex hormone-binding globulin) domain and the third EGF-like sequence. In addition, we observed several larger-scale events of alternative splicing that deleted multiple domains and were much less frequent than the canonical six sites of alternative splicing in neurexins. All of the six canonical events of alternative splicing appear to be independent of each other, suggesting that neurexins may exhibit an even larger isoform diversity than previously envisioned and comprise thousands of variants. Our data are consistent with the notion that a-neurexins represent extracellular protein-interaction scaffolds in which different LNS and EGF domains mediate distinct interactions that affect diverse functions and are independently regulated by independent events of alternative splicing.

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September 22, 2019  |  

PacBio sequencing and its applications.

Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing (SGS) technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation (SV) in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with diseases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Additionally, PacBio’s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

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September 22, 2019  |  

Emergence, retention and selection: A trilogy of origination for functional de novo proteins from ancestral lncRNAs in primates.

While some human-specific protein-coding genes have been proposed to originate from ancestral lncRNAs, the transition process remains poorly understood. Here we identified 64 hominoid-specific de novo genes and report a mechanism for the origination of functional de novo proteins from ancestral lncRNAs with precise splicing structures and specific tissue expression profiles. Whole-genome sequencing of dozens of rhesus macaque animals revealed that these lncRNAs are generally not more selectively constrained than other lncRNA loci. The existence of these newly-originated de novo proteins is also not beyond anticipation under neutral expectation, as they generally have longer theoretical lifespan than their current age, due to their GC-rich sequence property enabling stable ORFs with lower chance of non-sense mutations. Interestingly, although the emergence and retention of these de novo genes are likely driven by neutral forces, population genetics study in 67 human individuals and 82 macaque animals revealed signatures of purifying selection on these genes specifically in human population, indicating a proportion of these newly-originated proteins are already functional in human. We thus propose a mechanism for creation of functional de novo proteins from ancestral lncRNAs during the primate evolution, which may contribute to human-specific genetic novelties by taking advantage of existed genomic contexts.

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September 22, 2019  |  

The discovered chimeric protein plays the cohesive role to maintain scallop byssal root structural integrity.

Adhesion is essential for many marine sessile organisms. Unraveling the compositions and assembly of marine bioadheisves is the fundamental to understand their physiological roles. Despite the remarkable diversity of animal bioadhesion, our understanding of this biological process remains limited to only a few animal lineages, leaving the majority of lineages remain enigmatic. Our previous study demonstrated that scallop byssus had distinct protein composition and unusual assembly mechanism apart from mussels. Here a novel protein (Sbp9) was discovered from the key part of the byssus (byssal root), which contains two Calcium Binding Domain (CBD) and 49 tandem Epidermal Growth Factor-Like (EGFL) domain repeats. Modular architecture of Sbp9 represents a novel chimeric gene family resulting from a gene fusion event through the acquisition of CBD2 domain by tenascin like (TNL) gene from Na+/Ca2+ exchanger 1 (NCX1) gene. Finally, free thiols are present in Sbp9 and the results of a rescue assay indicated that Sbp9 likely plays the cohesive role for byssal root integrity. This study not only aids our understanding of byssus assembly but will also inspire biomimetic material design.

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September 22, 2019  |  

Predominant contribution of cis-regulatory divergence in the evolution of mouse alternative splicing.

Divergence of alternative splicing represents one of the major driving forces to shape phenotypic diversity during evolution. However, the extent to which these divergences could be explained by the evolving cis-regulatory versus trans-acting factors remains unresolved. To globally investigate the relative contributions of the two factors for the first time in mammals, we measured splicing difference between C57BL/6J and SPRET/EiJ mouse strains and allele-specific splicing pattern in their F1 hybrid. Out of 11,818 alternative splicing events expressed in the cultured fibroblast cells, we identified 796 with significant difference between the parental strains. After integrating allele-specific data from F1 hybrid, we demonstrated that these events could be predominately attributed to cis-regulatory variants, including those residing at and beyond canonical splicing sites. Contrary to previous observations in Drosophila, such predominant contribution was consistently observed across different types of alternative splicing. Further analysis of liver tissues from the same mouse strains and reanalysis of published datasets on other strains showed similar trends, implying in general the predominant contribution of cis-regulatory changes in the evolution of mouse alternative splicing. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

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