Dataset Information

RNA-Seq de novo assembly and differential transcriptome analysis of the nematode Ascaridia galli in relation to in vivo exposure to flubendazole.

ABSTRACT: The nematode Ascaridia galli (order Ascaridida) is an economically important intestinal parasite responsible for increased food consumption, reduced performance and elevated mortality in commercial poultry production. This roundworm is an emerging problem in several European countries on farms with laying hens, as a consequence of the recent European Union (EU) ban on conventional battery cages. As infection is associated with slow development of low levels of acquired protective immunity, parasite control relies on repeated use of dewormers (anthelmintics). Benzimidazoles (BZ) are currently the only anthelmintic registered in the EU for use in controlling A. galli and there is an obvious risk of overuse of one drug class, selecting for resistance. Thus we developed a reference transcriptome of A. galli to investigate the response in gene expression before and after exposure to the BZ drug flubendazole (FLBZ). Transcriptional variations between treated and untreated A. galli showed that transcripts annotated as mitochondrial glutamate dehydrogenase and cytochrome P450 were significantly down-regulated in treated worms, whereas transcripts homologous to heat shock proteins (HSP), catalase, phosphofructokinase, and a multidrug resistance P-glycoprotein (PGP1) were significantly up-regulated in treated worms. Investigation of candidate transcripts responsible for anthelmintic resistance in livestock nematodes led to identification of several tubulins, including six new isoforms of beta-tubulin, and several ligand-gated ionotropic receptors and ABC-transporters. We discovered several transcripts associated with drug binding and processing genes, but further characterisation using a larger set of worms exposed to BZs in functional assays is required to determine how these are involved in drug binding and metabolism.

SUBMITTER: Martis MM

PROVIDER: S-EPMC5669496 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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RNA-Seq de novo assembly and differential transcriptome analysis of the nematode Ascaridia galli in relation to in vivo exposure to flubendazole.

Martis Mihaela M MM Tarbiat Behdad B Tydén Eva E Jansson Désirée S DS Höglund Johan J

PloS one 20171103 11

The nematode Ascaridia galli (order Ascaridida) is an economically important intestinal parasite responsible for increased food consumption, reduced performance and elevated mortality in commercial poultry production. This roundworm is an emerging problem in several European countries on farms with laying hens, as a consequence of the recent European Union (EU) ban on conventional battery cages. As infection is associated with slow development of low levels of acquired protective immunity, paras ...[more]

PMID: 29099835

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Project description:BackgroundBarnyardgrass (Echinochloa crus-galli) is an important weed that is a menace to rice cultivation and production. Rapid evolution of herbicide resistance in this weed makes it one of the most difficult to manage using herbicides. Since genome-wide sequence data for barnyardgrass is limited, we sequenced the transcriptomes of susceptible and resistant barnyardgrass biotypes using the 454 GS-FLX platform.Results454 pyrosequencing generated 371,281 raw reads with an average length of 341.8 bp, which made a total length of 126.89 Mb (SRX160526). De novo assembly produced 10,142 contigs (∼5.92 Mb) with an average length of 583 bp and 68,940 singletons (∼22.13 Mb) with an average length of 321 bp. About 244,653 GO term assignments to the biological process, cellular component and molecular function categories were obtained. A total of 6,092 contigs and singletons with 2,515 enzyme commission numbers were assigned to 151 predicted KEGG metabolic pathways. Digital abundance analysis using Illumina sequencing identified 78,124 transcripts among susceptible, resistant, herbicide-treated susceptible and herbicide-treated resistant barnyardgrass biotypes. From these analyses, eight herbicide target-site gene groups and four non-target-site gene groups were identified in the resistant biotype. These could be potential candidate genes involved in the herbicide resistance of barnyardgrass and could be used for further functional genomics research. C4 photosynthesis genes including RbcS, RbcL, NADP-me and MDH with complete CDS were identified using PCR and RACE technology.ConclusionsThis is the first large-scale transcriptome sequencing of E. crus-galli performed using the 454 GS-FLX platform. Potential candidate genes involved in the evolution of herbicide resistance were identified from the assembled sequences. This transcriptome data may serve as a reference for further gene expression and functional genomics studies, and will facilitate the study of herbicide resistance at the molecular level in this species as well as other weeds.

Project description:BACKGROUND: RNA-seq has shown huge potential for phylogenomic inferences in non-model organisms. However, error, incompleteness, and redundant assembled transcripts for each gene in de novo assembly of short reads cause noise in analyses and a large amount of missing data in the aligned matrix. To address these problems, we compare de novo assemblies of paired end 90 bp RNA-seq reads using Oases, Trinity, Trans-ABySS and SOAPdenovo-Trans to transcripts from genome annotation of the model plant Ricinus communis. By doing so we evaluate strategies for optimizing total gene coverage and minimizing assembly chimeras and redundancy. RESULTS: We found that the frequency and structure of chimeras vary dramatically among different software packages. The differences were largely due to the number of trans-self chimeras that contain repeats in the opposite direction. More than half of the total chimeras in Oases and Trinity were trans-self chimeras. Within each package, we found a trade-off between maximizing reference coverage and minimizing redundancy and chimera rate. In order to reduce redundancy, we investigated three methods: 1) using cap3 and CD-HIT-EST to combine highly similar transcripts, 2) only retaining the transcript with the highest read coverage, or removing the transcript with the lowest read coverage for each subcomponent in Trinity, and 3) filtering Oases single k-mer assemblies by number of transcripts per locus and relative transcript length, and then finding the transcript with the highest read coverage. We then utilized results from blastx against model protein sequences to effectively remove trans chimeras. After optimization, seven assembly strategies among all four packages successfully assembled 42.9-47.1% of reference genes to more than 200 bp, with a chimera rate of 0.92-2.21%, and on average 1.8-3.1 transcripts per reference gene assembled. CONCLUSIONS: With rapidly improving sequencing and assembly tools, our study provides a framework to benchmark and optimize performance before choosing tools or parameter combinations for analyzing short-read RNA-seq data. Our study demonstrates that choice of assembly package, k-mer sizes, post-assembly redundancy-reduction and chimera cleanup, and strand-specific RNA-seq library preparation and assembly dramatically improves gene coverage by non-redundant and non-chimeric transcripts that are optimized for downstream phylogenomic analyses.

Dataset Information

RNA-Seq de novo assembly and differential transcriptome analysis of the nematode Ascaridia galli in relation to in vivo exposure to flubendazole.

Publications

RNA-Seq de novo assembly and differential transcriptome analysis of the nematode Ascaridia galli in relation to in vivo exposure to flubendazole.

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