Koglin Sven, Trense Daronja, Wink Michael, Sauer-Gürth Hedwig, Tietze Dieter Thomas
Institute for Pharmacy and Molecular Biotechnology, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany.
Zoological Institute and Museum, University of Greifswald, Greifswald, Germany.
PeerJ. 2017 Dec 13;5:e4045. doi: 10.7717/peerj.4045. eCollection 2017.
In recent years, next generation high throughput sequencing technologies have proven to be useful tools for investigations concerning the genomics or transcriptomics also of non-model species. Consequently, ornithologists have adopted these technologies and the respective bioinformatics tools to survey the genomes and transcriptomes of a few avian non-model species. The Common Blackbird is one of the most common bird species living in European cities, which has successfully colonized urban areas and for which no reference genome or transcriptome is publicly available. However, to target questions like genome wide gene expression analysis, a reference genome or transcriptome is needed.
Therefore, in this study two Common Blackbirds were sacrificed, their mRNA was isolated and analyzed by RNA-Seq to assemble a transcriptome and characterize it. Illumina reads (125 bp paired-end) and a Velvet/Oases pipeline led to 162,158 transcripts. For the annotation (using Blast+), an unfiltered protein database was used. SNPs were identified using SAMtools and BCFtools. Furthermore, mRNA from three single tissues (brain, heart and liver) of the same two Common Blackbirds were sequenced by Illumina (75 bp single-end reads). The draft transcriptome and the three single tissues were compared by their BLAST hits with the package VennDiagram in R.
Following the annotation against protein databases, we found evidence for 15,580 genes in the transcriptome (all well characterized hits after annotation). On 18% of the assembled transcripts, 144,742 SNPs were identified which are, consequently, 0.09% of all nucleotides in the assembled transcriptome. In the transcriptome and in the single tissues (brain, heart and liver), 10,182 shared genes were found.
Using a next-generation technology and bioinformatics tools, we made a first step towards the genomic investigation of the Common Blackbird. The assembled transcriptome is usable for downstream analyses such as differential gene expression analysis and SNP identification. This study shows the importance of the approach to sequence single tissues to understand functions of tissues, proteins and the phenotype.
近年来，新一代高通量测序技术已被证明是研究非模式生物基因组学或转录组学的有用工具。因此，鸟类学家采用了这些技术和相应的生物信息学工具来调查一些非模式鸟类物种的基因组和转录组。乌鸫是生活在欧洲城市中最常见的鸟类之一，它已成功地在城市地区定居，目前尚无公开可用的参考基因组或转录组。然而，要针对全基因组基因表达分析等问题，就需要参考基因组或转录组。
因此，在本研究中，处死了两只乌鸫，分离其mRNA并通过RNA测序进行分析，以组装转录组并对其进行表征。Illumina测序读数（125bp双端）和Velvet/Oases流程产生了162,158个转录本。对于注释（使用Blast+），使用了未过滤的蛋白质数据库。使用SAMtools和BCFtools鉴定单核苷酸多态性（SNP）。此外，对同两只乌鸫的三个单一组织（脑、心脏和肝脏）的mRNA进行Illumina测序（75bp单端读数）。通过R中的VennDiagram软件包，将转录组草图和三个单一组织的BLAST比对结果进行比较。
在与蛋白质数据库进行注释后，我们在转录组中发现了15,580个基因的证据（注释后所有特征明确的比对结果）。在18%的组装转录本上，鉴定出144,742个SNP，因此占组装转录组中所有核苷酸的0.09%。在转录组和单一组织（脑、心脏和肝脏）中，发现了10,182个共享基因。
利用下一代技术和生物信息学工具，我们朝着乌鸫的基因组研究迈出了第一步。组装的转录组可用于下游分析，如差异基因表达分析和SNP鉴定。本研究表明，对单一组织进行测序以了解组织、蛋白质和表型功能的方法具有重要意义。
