High-throughput sequencing of small RNAs in Persea americana

Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from Persea americana tissues (including leaves, flowers and fruit). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the maize genome under study. Small RNA libraries were derived from leaves, flowers and fruit of Persea americana. Total RNA was isolated using the Plant RNA Purification Reagent (Invitrogen), and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Doug Soltis for providing the plant material as well as Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods.

小分子RNA（small RNAs，21-24 nt）是调控基因表达的关键因子，可介导包括几乎所有植物在内的多种真核生物的转录及转录后基因沉默机制。
微RNA（miRNAs）与小干扰RNA（siRNAs）是两类主要的小分子RNA，二者在植物发育、胁迫响应及病原菌抗性中均发挥着已被证实的重要作用。
本研究采用深度测序技术（边合成边测序，Sequencing-By-Synthesis，简称SBS），构建了鳄梨（Persea americana）组织（含叶片、花和果实）中小分子RNA的序列资源。所得数据集的测序深度极高，使我们能够详细解析该物种中小分子RNA的多项关键特征，包括长度分布、组织特异性调控以及不同器官间的序列保守性。
我们还开发了数据库资源及专用网站（http://smallrna.udel.edu/），配套相关计算工具，以供其他研究者识别参与特定调控通路的新型微RNA或小干扰RNA，验证这些序列在其他植物物种中的保守程度，并将小分子RNA定位到研究中的玉米基因组的基因区域或更大基因组区段上。
小分子RNA文库源自鳄梨的叶片、花和果实组织。总RNA采用植物RNA纯化试剂（Invitrogen公司）提取，并提交至Illumina公司（美国加利福尼亚州海沃德市，http://www.illumina.com），参照Lu等人2007年报道的方法并略作修改，完成小分子RNA文库的构建。
该小分子RNA文库通过Illumina的边合成边测序（SBS）技术完成测序。
我们编写PERL脚本以去除接头序列，并统计每一种独特小分子RNA的表达丰度。
我们感谢Doug Soltis提供植物材料，同时感谢Kan Nobuta与Gayathri Mahalingam在计算方法方面提供的协助。