Variant linkage analysis using de novo transcriptome sequencing identifies a conserved phosphine resistance gene in insects
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PROVIDER: PRJEB24430 | ENA |
REPOSITORIES: ENA
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Project description:Next-generation sequencing methods enable identification of the genetic basis of traits in species that have no prior genomic information available. The combination of next-generation sequencing, variant analysis, and linkage is a powerful way of identifying candidate genes for a trait of interest. Here, we used a comparative transcriptomics [RNA sequencing (RNAseq)] and genetic linkage analysis approach to identify the rph1 gene. rph1 variants are responsible for resistance to the fumigant phosphine (PH3) that is used to control insect pests of stored grain. In each of the four major species of pest insect of grain we have investigated, there are two major resistance genes, rph1 and rph2, which interact synergistically to produce strongly phosphine-resistant insects. Using RNAseq and genetic linkage analyses, we identified candidate resistance (rph1) genes in phosphine-resistant strains of three species: Rhyzopertha dominica (129 candidates), Sitophilus oryzae (206 candidates), and Cryptolestes ferrugineus (645 candidates). We then compared these candidate genes to 17 candidate resistance genes previously mapped in Tribolium castaneum and found only one orthologous gene, a cytochrome b5 fatty acid desaturase (Cyt-b5-r), to be associated with the rph1 locus in all four species. This gene had either missense amino acid substitutions and/or insertion/deletions/frameshift variants in each of 18 phosphine-resistant strains that were not observed in the susceptible strains of the four species. We propose a model of phosphine action and resistance in which phosphine induces lipid peroxidation through reactive oxygen species generated by dihydrolipoamide dehydrogenase, whereas disruption of Cyt-b5-r in resistant insects decreases the polyunsaturated fatty acid content of membranes, thereby limiting the potential for lipid peroxidation.
| S-EPMC5937174 | biostudies-literature
Project description:Background:Spittle bugs and sharpshooters are well-known xylem sap-feeding insects and vectors of the phytopathogenic bacterium Xylella fastidiosa (Wells), a causal agent of Pierce's disease of grapevines and other crop diseases. Specialized feeding on nutrient-deficient xylem sap is relatively rare among insect herbivores, and only limited genomic and transcriptomic information has been generated for xylem-sap feeders. To develop a more comprehensive understanding of biochemical adaptations and symbiotic relationships that support survival on a nutritionally austere dietary source, transcriptome assemblies for three sharpshooter species and one spittlebug species were produced. Findings:Trinity-based de novo transcriptome assemblies were generated for all four xylem-sap feeders using raw sequencing data originating from whole-insect preps. Total transcripts for each species ranged from 91?384 for Cuerna arida to 106?998 for Homalodisca liturata with transcript totals for Graphocephala atropunctata and the spittlebug Clastoptera arizonana falling in between. The percentage of transcripts comprising complete open reading frames ranged from 60% for H. liturata to 82% for C. arizonana. Bench-marking universal single-copy orthologs analyses for each dataset indicated quality assemblies and a high degree of completeness for all four species. Conclusions:These four transcriptomes represent a significant expansion of data for insect herbivores that feed exclusively on xylem sap, a nutritionally deficient dietary source relative to other plant tissues and fluids. Comparison of transcriptome data with insect herbivores that utilize other dietary sources may illuminate fundamental differences in the biochemistry of dietary specialization.
| S-EPMC5467018 | biostudies-literature
Project description:Obesity has emerged as a formidable health crisis due to its association with metabolic risk factors such as diabetes, dyslipidaemia and hypertension. Recent work has demonstrated the multifaceted roles of lncRNAs in regulating mouse adipose development, but its implication in human adipocytes remain largely unknown at least partially due to the lack of a comprehensive lncRNA catalog, particularly those specifically expressed in brown adipose tissue (BAT). In this study, we performed deep RNA-seq on adult subcutaneous, omental and fetal brown adipose tissues to de novo construct a catalog of 3,149 adipose active lncRNAs of which 1,351 are specifically detected in BAT. We further identified 318 lncRNAs conserved between human and mouse which, compared with non-conserved ones, are more broadly expressed in multiple cell types. One of these, lnc-dPRDM16, is transcribed divergently from Prdm16, tightly correlated with Prdm16 (R ≥ 0.7) in both mouse and human, and co-expressed (R ≥ 0.7) with protein-coding genes enriched in lipid and fatty acid catabolic processes. Loss of function of lnc-dPRDM16 led to a down-regulation of Prdm16 and an obvious reduction of adipogenesis in brown adipocyte culture. Together, our work has provided a comprehensive human adipose catalog built from diverse fat types, which when applied to our roadmap, identifies lnc-dPRDM16 as a promising modulator of adipose development for future clinical research.
2018-04-16 | GSE97205 | GEO
Project description:The accuracy of sequencing single DNA molecules with nanopores is continually improving, but de novo genome sequencing and assembly using only nanopore data remain challenging. Here we describe PoreSeq, an algorithm that identifies and corrects errors in nanopore sequencing data and improves the accuracy of de novo genome assembly with increasing coverage depth. The approach relies on modeling the possible sources of uncertainty that occur as DNA transits through the nanopore and finds the sequence that best explains multiple reads of the same region. PoreSeq increases nanopore sequencing read accuracy of M13 bacteriophage DNA from 85% to 99% at 100× coverage. We also use the algorithm to assemble Escherichia coli with 30× coverage and the ? genome at a range of coverages from 3× to 50×. Additionally, we classify sequence variants at an order of magnitude lower coverage than is possible with existing methods.
| S-EPMC4877053 | biostudies-literature
Project description:The de novo ceramide synthesis pathway is essential to human biology and health, but genetic influences remain unexplored. The core function of this pathway is the generation of biologically active ceramide from its precursor, dihydroceramide. Dihydroceramides have diverse, often protective, biological roles; conversely, increased ceramide levels are biomarkers of complex disease. To explore the genetics of the ceramide synthesis pathway, we searched for deleterious nonsynonymous variants in the genomes of 1,020 Mexican Americans from extended pedigrees. We identified a Hispanic ancestry-specific rare functional variant, L175Q, in delta 4-desaturase, sphingolipid 1 (DEGS1), a key enzyme in the pathway that converts dihydroceramide to ceramide. This amino acid change was significantly associated with large increases in plasma dihydroceramides. Indexes of DEGS1 enzymatic activity were dramatically reduced in heterozygotes. CRISPR/Cas9 genome editing of HepG2 cells confirmed that the L175Q variant results in a partial loss of function for the DEGS1 enzyme. Understanding the biological role of DEGS1 variants, such as L175Q, in ceramide synthesis may improve the understanding of metabolic-related disorders and spur ongoing research of drug targets along this pathway.
| S-EPMC6718439 | biostudies-literature
Project description:Kinetochores mediate chromosome attachment to the mitotic spindle to ensure accurate chromosome segregation. Budding yeast is an excellent organism for kinetochore assembly studies because it has a simple defined centromere sequence responsible for the localization of >65 proteins. In addition, yeast is the only organism where a conditional centromere is available to allow studies of de novo kinetochore assembly. Using a conditional centromere, we found that yeast kinetochore assembly is not temporally restricted and can occur in both G1 phase and prometaphase. We performed the first investigation of kinetochore assembly in the absence of the centromeric histone H3 variant Cse4 and found that all proteins tested depend on Cse4 to localize. Consistent with this observation, Cse4-depleted cells had severe chromosome segregation defects. We therefore propose that yeast kinetochore assembly requires both centromeric DNA specificity and centromeric chromatin.
| S-EPMC1289410 | biostudies-literature
Project description:DNA methylation plays a critical role in controlling states of gene activity in most eukaryotic organisms, and it is essential for proper growth and development. Patterns of methylation are established by de novo methyltransferases and maintained by maintenance methyltransferase activities. The Dnmt3 family of de novo DNA methyltransferases has recently been characterized in animals. Here we describe DNA methyltransferase genes from both Arabidopsis and maize that show a high level of sequence similarity to Dnmt3, suggesting that they encode plant de novo methyltransferases. Relative to all known eukaryotic methyltransferases, these plant proteins contain a novel arrangement of the motifs required for DNA methyltransferase catalytic activity. The N termini of these methyltransferases contain a series of ubiquitin-associated (UBA) domains. UBA domains are found in several ubiquitin pathway proteins and in DNA repair enzymes such as Rad23, and they may be involved in ubiquitin binding. The presence of UBA domains provides a possible link between DNA methylation and ubiquitin/proteasome pathways.
| S-EPMC18343 | biostudies-literature
Project description:The main genetic factors for familial melanoma remain unknown in more than 75% of families. CDKN2A is mutated in around 20% of melanoma-prone families. Other high-risk melanoma susceptibility genes explain less than 3% of families studied to date. We performed the first genome-wide linkage analysis in CDKN2A-negative Spanish melanoma-prone families to identify novel melanoma susceptibility loci. We included 68 individuals from 2, 3 and 6 families with 2, 3 and at least 4 melanoma cases. We detected a locus with significant linkage evidence at 11q14.1-q14.3, with a maximum het-TLOD of 3.449 (rs12285365:A>G), using evidence from multiple pedigrees. The genes contained by the subregion with the strongest linkage evidence were: DLG2, PRSS23, FZD4 and TMEM135. We also detected several regions with suggestive linkage evidence (TLOD>1.9) (1q, 6p, 7p, 11q, 12p, 13q) including the region previously detected in melanoma-prone families from Sweden at 3q29. The family specific analysis revealed three loci with suggestive linkage evidence for family #1: 1q31.1-q32.1 (max. TLOD 2.447), 6p24.3-p22.3 (max. TLOD 2.409) and 11q13.3-q21 (max. TLOD 2.654). Future next generation sequencing studies of these regions may allow the identification of new melanoma susceptibility genetic factors.
2019-01-01 | GSE109206 | GEO
Project description:The main genetic factors for familial melanoma remain unknown in more than 75% of families. CDKN2A is mutated in around 20% of melanoma-prone families. Other high-risk melanoma susceptibility genes explain less than 3% of families studied to date. We performed the first genome-wide linkage analysis in CDKN2A-negative Spanish melanoma-prone families to identify novel melanoma susceptibility loci. We included 68 individuals from 2, 3 and 6 families with 2, 3 and at least 4 melanoma cases. We detected a locus with significant linkage evidence at 11q14.1-q14.3, with a maximum het-TLOD of 3.449 (rs12285365:A>G), using evidence from multiple pedigrees. The genes contained by the subregion with the strongest linkage evidence were: DLG2, PRSS23, FZD4 and TMEM135. We also detected several regions with suggestive linkage evidence (TLOD>1.9) (1q, 6p, 7p, 11q, 12p, 13q) including the region previously detected in melanoma-prone families from Sweden at 3q29. The family specific analysis revealed three loci with suggestive linkage evidence for family #1: 1q31.1-q32.1 (max. TLOD 2.447), 6p24.3-p22.3 (max. TLOD 2.409) and 11q13.3-q21 (max. TLOD 2.654). Future next generation sequencing studies of these regions may allow the identification of new melanoma susceptibility genetic factors.
2019-01-01 | GSE109207 | GEO
Project description:BACKGROUND:Otocephaly is a rare lethal malformation of the first branchial arch. While the knowledge on the causes of otocephaly in animals is limited, different syndromic forms in man are associated with variants of the PRRX1 and OTX2 genes. CASE PRESENTATION:A stillborn male lamb of the Istrian Pramenka sheep breed showed several congenital craniofacial anomalies including microstomia, agnathia, aglossia, and synotia. In addition, the lamb had a cleft palate, a small opening in the ventral neck region, a cystic oesophagus and two hepatic cysts. The brain was normally developed despite the deformed shape of the head. Taken together the findings led to a diagnosis of otocephaly. Whole-genome sequencing was performed from DNA of the affected lamb and both parents revealing a heterozygous single nucleotide variant in the OTX2 gene (Chr7: 71478714G?>?A). The variant was absent in both parents and therefore due to a de novo mutation event. It was a nonsense variant, XM_015097088.2:c.265C?>?T; which leads to an early premature stop codon and is predicted to truncate more than 70% of the OTX2 open reading frame (p.Arg89*). CONCLUSIONS:The genetic findings were consistent with the diagnosis of the otocephaly and provide strong evidence that the identified loss-of-function variant is pathogenic due to OTX2 haploinsufficiency. The benefits of trio-based whole-genome sequencing as an emerging tool in veterinary pathology to confirm diagnosis are highlighted.
| S-EPMC6977343 | biostudies-literature