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，我们采用了一种新的成功的方法来分析高通量RNA测序(RNA-seq)中的基因表达。植物中选择性剪接(AS)的高发生率(在含有内含子的基因中发现600%)需要方法来区分和量化为变异体或异构体。我们采用了科学家在人类癌症中分析基因表达和AS的方法。这种方法使用了像SAMMON这样的程序，它不需要将阅读映射到基因组，而是使用参考转录数据集来量化转录。我们已经为拟南芥生成了RTD(Zhang等人，2015,2016)，其中涉及开发内部管道，以消除RTD中的冗余。使用这个系统和从常温到低温转换的植物的一段时间的RNA-seq数据，已经证明了转录特异的表达，包括作为对冷的反应，并发现了参与冷反应的新基因。博士项目将开发用于在农作物(土豆和大麦)中构建RTD的计算管道，并将使用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。产生特定转录本和特定等位基因的表达数据的能力将极大地增强我们分析基因表达和识别植物/作物过程中的关键基因的能力，例如非生物和生物胁迫反应。

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.