Construction and refinement of Reference Transcript Dataset annotations for fast and accurate transcript quantification in barley and potato

在模式植物拟南芥中，我们已经采取了一种新的和成功的方法来分析基因表达的高通量RNA测序（RNA-seq）。植物中选择性剪接（AS）的高发生率（在> 600%的含内含子基因中发现）需要区分和定量AS变体或同种型的方法。我们采用了科学家分析人类癌症中基因表达和AS的方法。该方法使用像SALMON这样的程序，它不需要将读数映射到基因组，而是使用参考转录数据集来量化转录本。我们已经产生了拟南芥的RTD（Zhang et al.，2015年，2016年），其中涉及内部管道的开发，以消除RTD中的冗余。使用该系统与来自从正常温度转移到低温温度的植物的时间过程的RNA-seq数据已经证明了转录物特异性表达，包括AS对冷的响应，并鉴定了参与冷响应的新基因。博士项目将开发用于构建作物（马铃薯和大麦）RTD的计算管道，这些管道将使用RNA-seq数据集进行测试。这将需要为管道和各种下游分析开发算法。产生转录本特异性和等位基因特异性表达数据的能力将大大提高我们分析基因表达和鉴定植物/作物过程（如非生物和生物胁迫响应）中关键基因的能力。

In the model plant, Arabidopsis, we have taken a new and successful approach to analyse gene expression from high-throughput sequencing of RNA (RNA-seq). The high incidence of alternative splicing (AS) in plants (found in >600% of intron-containing genes) requires methods to distinguish and quantify AS variants or isoforms. We have taken an approach used by scientists analyzing gene expression and AS in human cancers. The approach uses programmes like SALMON, which does not require mapping of reads to genomes but instead use a Reference Transcript Dataset to quantify transcripts. We have generated an RTD for Arabidopsis (Zhang et al., 2015, 2016), which involved the development of in-house pipelines for removing redundancy in the RTD. Using this system with RNA-seq data from a time-course of plants transferred from normal to cold temperatures has already demonstrated transcript-specific expression including AS responses to cold, and identified new genes involved in the cold response. The PhD project will develop computational pipelines for construction of RTDs in crop plants (potato and barley) which will be tested with RNA-seq datasets. This will require development of algorithms for the pipelines and for various downstream analyses. The ability to generate transcript-specific and allele-specific expression data will greatly enhance our ability to analyse gene expression and identify key genes in plant/crop processes such as abiotic and biotic stress responses.