Identification of alternatively spliced gene isoforms and novel noncoding RNAs by single-molecule long-read sequencing in Camellia
Identification of alternatively spliced gene isoforms and novel noncoding RNAs by single-molecule long-read sequencing in Camellia
复制标题
通过单分子长读长测序鉴定山茶花中可变剪接基因异构体和新型非编码 RNA
DOI：
10.1080/15476286.2020.1738703
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发表时间：
2020-07-02
期刊：
影响因子：
4.1
通讯作者：
Yin, Hengfu
中科院分区：
文献类型：
--
作者：
Hu, Zhikang;Lyu, Tao;Yin, Hengfu
摘要对全长转录本进行直接单分子测序可以有效地鉴定基因异构体，这容易进行选择性剪接(AS)、多聚腺苷酸化和长时间的非编码RNA分析。然而，识别具有新的调节功能的基因异构体和长的非编码RNA仍然具有挑战性，特别是对于没有参考基因组的物种。在这里，我们提出了一个综合分析长读和短读相结合的茶树转录组测序。通过一个新的反向追踪分裂位点的生物信息学管道，我们从61,838个转录本中发现了257,692个AS位点；通过比对短读数，我们发现了13,068个AS异构体。我们已经确定了组织特异性的异构体以及6,373个在所有组织中发现的事件。此外，我们还分析了转录本的多聚腺苷酸化(PolyA)模式，发现AS转录本和非AS转录本对PolyA信号的偏好不同。此外，我们还通过转录组和小RNA测序的综合分析预测了阶段性小干扰RNA(PhasiRNA)基因座。我们已经证明，一个新进化的来自脂肪氧合酶的phasiRNA基因座产生了12个连续的21bp的次级RNAs，这些RNAs对山茶花的冷热胁迫做出了响应。我们对异构体转录组的研究提供了对基因剪接和功能的洞察，这可能有助于从机制上理解植物。
ABSTRACT Direct single-molecule sequencing of full-length transcripts allows efficient identification of gene isoforms, which is apt to alternative splicing (AS), polyadenylation, and long non-coding RNA analyses. However, the identification of gene isoforms and long non-coding RNAs with novel regulatory functions remains challenging, especially for species without a reference genome. Here, we present a comprehensive analysis of a combined long-read and short-read transcriptome sequencing in Camellia japonica. Through a novel bioinformatic pipeline of reverse-tracing the split-sites, we have uncovered 257,692 AS sites from 61,838 transcripts; and 13,068 AS isoforms have been validated by aligning the short reads. We have identified the tissue-specific AS isoforms along with 6,373 AS events that were found in all tissues. Furthermore, we have analysed the polyadenylation (polyA) patterns of transcripts, and found that the preference for polyA signals was different between the AS and non-AS transcripts. Moreover, we have predicted the phased small interfering RNA (phasiRNA) loci through integrative analyses of transcriptome and small RNA sequencing. We have shown that a newly evolved phasiRNA locus from lipoxygenases generated 12 consecutive 21 bp secondary RNAs, which were responsive to cold and heat stress in Camellia. Our studies of the isoform transcriptome provide insights into gene splicing and functions that may facilitate the mechanistic understanding of plants.