Integrated single-molecule long-read sequencing and Illumina sequencing reveal the resistance mechanism of Psathyrostachys huashanica in response to barley yellow dwarf virus-GAV
结合单分子长读测序和Illumina测序，揭示了华山百合对大麦黄矮病毒gav的抗性机制
Transcriptomics is the study of gene structure, expression, and regulation. Short-read second-generation sequencing (SGS) has become a powerful tool for quantifying gene expression levels and exploring transcriptional gene regulation (Buermans and den Dunnen 2014). However, SGS has its limitations, such as short read lengths, which makes it poorly suited for the assembly of complex genomes and transcriptomes as well as full-length isoforms and methylation detection (Rhoads and Au 2015; An et al. 2018). Single-molecule real-time (SMRT) sequencing, a form of third-generation sequencing, was developed by Pacific Biosciences (PacBio) and offers an alternative approach to overcome these limitations, enhancing our understanding of the transcriptome complexity (Rhoads and Au 2015; An et al. 2018).
Full-length cDNA sequencing is fundamental to structural and functional genomics studies, and is used for genome annotation, the identification of novel genes and isoforms, as well as the characterization of long non-coding RNAs (lncRNAs), especially those without a reference genome (Roberts et al. 2013; Liu et al. 2017a). Additionally, SMRT sequencing is used for the assembly of plant genomes based on its long reads, such as its application in Triticum aestivum (Zimin et al. 2017a), loblolly pine (Zimin et al. 2017b), Zea mays (Jiao et al. 2017) and rubber tree (Pootakham et al. 2017). On the other hand, full-length cDNA sequencing has been combined with Illumina-based RNA-seq datasets in Sorghum bicolor (Abdel-Ghany et al. 2016), Zea mays (Wang et al. 2016), cotton (Wang et al. 2018), sugarcane (Hoang et al. 2017), coffee bean (Cheng et al. 2017) and sweet potato (Luo et al. 2017), to characterize the complexity of transcriptomes. Moreover, SMRT sequencing has also been widely used to construct transcriptomes in some plants without a reference genome, such as Camellia sinensis (Xu et al. 2017), Astragalus membranaceus (Li et al. 2017a), and Halogeton glomeratus (Wang et al. 2017), but it has not been used in P. huashanica.
In this study, we constructed full-length cDNA libraries of P. huashanica and performed SMRT sequencing to generate large-scale full-length transcripts. Then, we used full-length transcripts as a reference to identify differentially-expressed genes (DEGs) in P. huashanica in response to BYDV-GAV infection and found plenty of defense-related genes induced by the viral invasion. Our results provide novel insights into the resistance response of P. huashanica to BYDV-GAV infection and may contribute to the utilization of resistance resources of P. huashanica in the future.
The PacBio full-length isoform sequencing platform can be used to obtain transcripts without assembly to overcome the difficulty of short-reads in next-generation sequencing (Bayega et al. 2018). However, even though PacBio offers longer reads than other current platforms, it has a higher error rate (Au et al. 2012). The short-reads from the Illumina platform are widely used for RNA-seq differential gene expression analysis since it provides sufficient depth and a lower error rate compared to reads generated from PacBio (Buermans and den Dunnen 2014). Therefore, we used clean reads created from short-read next-generation sequencing (NGS) technology to correct the PacBio transcript isoforms (Mahmoud et al. 2017).
转录组学是研究基因结构、表达和调控的学科。
短读二代测序(SGS)已经成为量化基因表达水平和探索转录基因调控的有力工具(Buermans和den Dunnen 2014)。
然而，SGS也有其局限性，如read长度短，这使得它不适合复杂基因组和转录组的装配，以及全长亚型和甲基化检测(Rhoads和Au 2015;
An等(2018)。
由太平洋生物科学公司(PacBio)开发的第三代测序单分子实时测序(SMRT)为克服这些局限性提供了一种替代方法，增强了我们对转录组复杂性的理解(Rhoads和Au 2015;
An等(2018)。
全长cDNA测序是结构和功能基因组学研究的基础，用于基因组注释、新基因和异构体的识别，以及长非编码rna (lncrna)的表征，特别是那些没有参考基因组的lncrna (Roberts et al. 2013;
Liu等，2017a)。
此外，SMRT测序还用于基于长序列的植物基因组组装，如在小麦(Zimin et al. 2017a)、红叶松(Zimin et al. 2017b)、玉米(Jiao et al. 2017)和橡橡胶树(Pootakham et al. 2017)中的应用。
另一方面,全长cDNA序列结合Illumina-based RNA-seq数据集在高粱二色的(Abdel-Ghany et al . 2016),玉蜀黍(王et al . 2016),棉花(王et al . 2018),甘蔗(黄平君et al . 2017),咖啡豆(Cheng et al . 2017年)和甘薯(罗et al . 2017),描述转录组的复杂性。
此外，SMRT测序也被广泛应用于一些没有参考基因组的植物中构建转录组，如山茶(Xu et al. 2017)、黄芪(Li et al. 2017a)、肾小素(Halogeton肾癌(Wang et al. 2017)等，但尚未在华山茶中应用。
在本研究中，我们构建了华山楝的全长cDNA文库，并进行了SMRT测序以获得大规模的全长转录本。
然后，我们利用全长转录本作为参考，鉴定了华山莲对BYDV-GAV感染的差异表达基因(DEGs)，发现了大量由病毒入侵诱导的防御相关基因。
本研究结果为了解华山合胞菌对BYDV-GAV感染的抗性反应提供了新的思路，对今后华山合胞菌的抗性资源利用有一定的参考价值。
PacBio全长isoform测序平台可以在不经过组装的情况下获得转录本，以克服下一代测序短读的困难(Bayega et al. 2018)。
然而，尽管PacBio提供的读取时间比目前其他平台更长，但它的错误率也更高(Au et al. 2012)。
Illumina平台的短reads被广泛用于RNA-seq差异基因表达分析，因为与PacBio产生的reads相比，该短reads具有足够的深度和较低的错误率(Buermans和den Dunnen 2014)。
因此，我们使用short-read下一代测序(NGS)技术创建的clean reads来校正PacBio转录子亚型(Mahmoud et al. 2017)。