Abstract
BackgroundIn 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.MethodsTherefore, in this study two Common Blackbirds were sacrificed, their mRNA was isolated and analyzed by RNA-Seq to de novo 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.ResultsFollowing 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.DiscussionUsing a next-generation technology and bioinformatics tools, we made a first step towards the genomic investigation of the Common Blackbird. The de novo 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.
Highlights
High-throughput or next-generation sequencing (NGS) is a useful tool for organisms with established reference genomes and transcriptomes, and for creating reference transcriptomes and genomes for non-model organisms
The sequencing of the pooled Blackbird mRNA led to 48,898,319 PE reads for the urban individual and to 68,085,209 PE reads for the rural Blackbird
Using BLAST+ with the unfiltered database, 48,607 (29.98%) of all contigs could be annotated to entries in the protein database, but 11.88% of those contigs were annotated to
Summary
High-throughput or next-generation sequencing (NGS) is a useful tool for organisms with established reference genomes and transcriptomes, and for creating reference transcriptomes and genomes for non-model organisms. With the still ongoing development of NGS technologies and with the help of bioinformatics tools for the analysis of the resulting large data sets, numerous publications concerning avian transcriptome or genome characterizations have already been published (Lerner & Fleischer, 2010; Basch et al, 2011; Botero-Castro et al, 2013; Shanks et al, 2013; Kraus & Wink, 2015; Xu et al, 2016). Generation high throughput sequencing technologies have proven to be useful tools for investigations concerning the genomics or transcriptomics of non-model species. Ornithologists have adopted these technologies and the respective bioinformatics tools to survey the genomes and transcriptomes of a few avian non-model species. This study shows the importance of the approach to sequence single tissues to understand functions of tissues, proteins and the phenotype
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