定向RNA-Seq技术在铜绿假单胞菌的应用

Strand-specific deep sequencing of transcriptome in Pseudomonas aeruginosa

目的探讨定向转录组测序技术在铜绿假单胞菌的应用,为研究基因组的注释和表达调控提供重要手段。方法抽提菌体RNA并去除rRNA,连接接头后用特异性引物反转录成cDNA,PCR扩增后测序;所得数据采用Bowtie和rSeq软件分析。结果在所有26 557 654条读段中,除去无效读段209 928条,与基因组匹配的读段为26 347 726条,测序结果能覆盖几乎99%的基因组,78.7%的基因来源于两个方向转录本映射读段,读段数最高的基因是ssrA,发现新的基因2 283个。结论成功建立了铜绿假单胞菌中的定向RNA-Seq技术。

Objective To provide the effective strategy for genome annotation and transcription regulation by studying the strand- specific deep sequencing of transcriptome in Pseudomonas aeruginosa. Methods RNA was extracted from Pseudomonas aeruginosa PAO1. After rRNA was removed, the RNA was inversely transcribed into cDNA with specific primer and sequenced by PCR amplification. The data were analyzed using Bowtie and rSeq software. Results Of the 26,557,654 trimmed read fragments （except for 209,928 no-valid read fragments）, 26,347,726 were the genome matching read fragments, indicating that the sequencing could cover almost 99% of the genomes. Seventy-eight point seven percent of the genome were the mapped reads on both strands. The most highly represented gene was ssrA and 2 283 new annotation genes were discovered. Conclusion Strand-specific deep sequencing of transcriptome is successful in Pseudomonas aeruginosa.