Project description:Accurate annotations of genes and their transcripts is a foundation of genomics, but no annotation technique presently combines throughput and accuracy. As a result, the GENCODE reference collection of long noncoding RNAs remains far from complete: many are fragmentary, while thousands more remain uncatalogued. To accelerate lncRNA annotation, we have developed RNA Capture Long Seq (CLS), combining targeted RNA capture with third generation long-read sequencing. We present an experimental re-annotation of the entire GENCODE intergenic lncRNA populations in matched human and mouse tissues. CLS approximately doubles the complexity of targeted loci, both in terms of validated splice junctions and transcript models. Through its identification of full-length transcript models, CLS allows the first definitive measurement of promoter features, gene structure and protein-coding potential of lncRNAs. Thus CLS removes a longstanding bottleneck of transcriptome annotation, generating manual-quality full-length transcript models at high-throughput scales.
Project description:Long non-coding RNAs (lncRNA) constitute a large fraction of mammalian transcriptomes that still remains unexplored, mainly due to the lack of comprehensive, high-quality lncRNA annotation that limits the possibility to fully explore their functional capacity. We have developed RACE-seq, an experimental workflow based on RACE (Rapid Amplification of cDNA Ends) and long read RNA sequencing, aimed at both rare isoform discovery and better definition of gene boundaries. We applied 3â and 5â RACE-seq on 398 low-expressed GENCODE v7 lncRNA genes in seven human tissues (brain, testis, heart, kidney, liver, lung and spleen). The sequences obtained led to the discovery of 2,641 on-target, previously unknown alternative transcripts. Novel isoforms extended 60% of the 398 targeted lncRNA loci further in either 5' or 3', and often reached genome hallmarks typical of gene boundaries. In parallel, we used nested RACE-seq, and confirmed that nested RACE-seq has overwhelmingly better sensitivity than its standard counterpart.
Project description:Heat stress is a major limiting factor for grain yield and grain quality in wheat production. In crops, abiotic stresses have transgenerational effects and the mechanistic basis of stress memory is associated with epigenetic regulation. The current study presents the first systematic analysis of the transgenerational effects of post-anthesis heat stress in tetraploid wheat. Genotype-dependent response patterns to parental and progeny heat stress were found for the leaf physiological traits, harvest components, and grain quality traits measured. Parental heat stress had positive influence on the offspring under re-occurring stress for traits like chlorophyll content, grain weight, grain number and grain total starch content. Integrated sequencing analysis of the small RNAome, mRNA transcriptome, and mRNA degradome provided the first description of the molecular networks mediating heat stress adaption under transgenerational influence. The expression profile of 1771 microRNAs (733 being novel) and 66,559 genes was provided, with differentially expressed microRNAs and genes identified subject to the progeny treatment, parental treatment and tissue type factors. Gene Ontology and KEGG pathway annotation of stress responsive microRNAs-mRNA modules provided further information on their functional roles in biological processes like hormone homeostasis, signal transduction, and protein stabilization. Our results provide new sights on the molecular basis of transgenerational heat stress adaptation, which can be used for improving thermos-tolerance in breeding.
Project description:High temperature during the grain-filling stage causes deleterious effects on storage material accumulation and grain quality. But it is still unclear how high temperature affects storage materials accumulation. In this study, we systemically analyzed the expression pattern of rice genes under high temperture during the grain-filling stage.