USDA-ARS Plant Science Research Unit and Department of Agronomy and Plant Genetics, University of Minnesota, St Paul, MN 55108, USA.
BMC Genomics. 2013 Jul 11;14:471. doi: 10.1186/1471-2164-14-471.
Next generation sequencing provides new opportunities to explore transcriptomes. However, challenges remain for accurate differentiation of homoeoalleles and paralogs, particularly in polyploid organisms with no supporting genome sequence. In this study, RNA-Seq was employed to generate and characterize the first gene expression atlas for hexaploid oat.
The software packages Trinity and Oases were used to produce a transcript assembly from nearly 134 million 100-bp paired-end reads from developing oat seeds. Based on the quality-parameters employed, Oases assemblies were superior. The Oases 67-kmer assembly, denoted dnOST (de novo Oat Seed Transcriptome), is over 55 million nucleotides in length and the average transcript length is 1,043 nucleotides. The 74.8× sequencing depth was adequate to differentiate a large proportion of putative homoeoalleles and paralogs. To assess the robustness of dnOST, we successfully identified gene transcripts associated with the biosynthetic pathways of three compounds with health-promoting properties (avenanthramides, tocols, β-glucans), and quantified their expression.
To our knowledge, this study provides the first direct performance comparison between two major assemblers in a polyploid organism. The workflow we developed provides a useful guide for comparable analyses in other organisms. The transcript assembly developed here is a major advance. It expands the number of oat ESTs 3-fold, and constitutes the first comprehensive transcriptome study in oat. This resource will be a useful new tool both for analysis of genes relevant to nutritional enhancement of oat, and for improvement of this crop in general.
下一代测序为探索转录组提供了新的机会。然而，在没有支持基因组序列的多倍体生物中，同源等位基因和旁系同源物的准确区分仍然存在挑战。在这项研究中，RNA-Seq 被用于生成和描述六倍体燕麦的第一个基因表达图谱。
使用 Trinity 和 Oases 软件包从发育中的燕麦种子的近 1.34 亿个 100bp 配对末端读取中产生转录体组装。基于所采用的质量参数，Oases 组装更好。Oases 67-kmer 组装，称为 dnOST（从头燕麦种子转录组），长度超过 5500 万个核苷酸，平均转录本长度为 1043 个核苷酸。74.8×测序深度足以区分大部分假定的同源等位基因和旁系同源物。为了评估 dnOST 的稳健性，我们成功鉴定了与三种具有促进健康特性的化合物（燕麦蒽酰胺、生育酚、β-葡聚糖）生物合成途径相关的基因转录本，并对其表达进行了定量。
据我们所知，这项研究在多倍体生物中首次直接比较了两种主要的组装器。我们开发的工作流程为其他生物中的类似分析提供了有用的指南。这里开发的转录本组装是一个重大进展。它将燕麦 EST 的数量扩大了 3 倍，并构成了燕麦的第一个综合转录组研究。该资源将成为分析与燕麦营养增强相关的基因以及总体上改进这种作物的有用新工具。
