Complete transcriptome of bir-2 knockdown C. elegans nematodes with and without exposure to pathogenic bacteria Enterococcus faecalis.

Purpose: The goals of this study are to examine the role of C. elegans gene bir-2 in innate immunity. The method is to compare differences in gene expression of transcriptomes of C. elegans in the presence and absence of a bir-2 knockdown and with and without pathogenic bacteria. Results: We mapped about 4 million sequence paired-end reads of 251 nucleotide length for each of the 16 samples, trimmed the reads with Trimmomatic (v.0.38), mapped to the WBGene C. elegans genome (build WBcel235.99) using HISAT2 (v.2.1.0), assembled and merged transcripts with StringTie. SamTools were used to convert the BAM files. Differential expression was evaluated using DESEQ2 in R. Approximately 4% of the transcripts showed differential expression between the WT and bir-2 knockdown, with a fold change ?2 and p value <0.05.!Series_summary = Methods: C. elegans were grown, in quadruplicate, exposed to HT115 bacteria expressing RNAi for bir-2 with and without E. faecalis exposure. Total RNA was purified and Illumina poly-A selection, library construction (KAPA RNA Hyperprep) and deep sequencing, using Illumina HiSeq2000 at UNH Hubbard Genome Center. The sequence reads were passed through FASTQC and Trimmomatic, and were analyzed at the transcript isoform level. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Overall design: Whole animal mRNA profiles of C. elegans nematodes +/- bir-2 knockdown +/- E. faecalis

研究目的：本研究旨在探究秀丽隐杆线虫（C. elegans）基因bir-2在先天免疫中的作用。实验方法为对比秀丽隐杆线虫在存在/不存在bir-2基因敲降、以及存在/不存在致病菌时的转录组基因表达差异。
实验结果：我们为16个样本各获取了约400万条长度为251核苷酸的双端测序序列reads，使用Trimmomatic（v.0.38）对reads进行质控修剪，通过HISAT2（v.2.1.0）将序列比对至WBGene秀丽隐杆线虫参考基因组（版本WBcel235.99），再利用StringTie完成转录本的组装与合并。使用SamTools转换BAM格式文件，通过R语言中的DESEQ2进行差异表达分析。结果显示，约4%的转录本在野生型（WT）与bir-2敲降组间存在差异表达，其倍数变化≥2且p值<0.05。
!系列摘要 = 实验方法：将秀丽隐杆线虫以四重复形式培养，分别用表达bir-2 RNAi的HT115细菌进行处理，并辅以/不辅以粪肠球菌（E. faecalis）暴露。提取总RNA后，采用Illumina poly-A富集、KAPA RNA Hyperprep试剂盒构建文库，并于新罕布什尔大学哈伯德基因组中心（UNH Hubbard Genome Center）使用Illumina HiSeq2000进行深度测序。对测序reads进行FASTQC质控与Trimmomatic修剪，并在转录异构体水平开展分析。
研究结论：本研究首次借助RNA测序（RNA-seq）技术，对具有生物学重复的秀丽隐杆线虫视网膜转录组进行了详细分析。本研究报道的优化数据分析流程，可为表达谱的比较研究提供参考框架。研究结果表明，下一代测序（Next-Generation Sequencing，NGS）技术能够对细胞或组织内的mRNA含量实现全面且更为精准的定量与定性评估。综上，基于RNA测序的转录组表征能够加速遗传网络分析，并助力解析复杂的生物学功能。
整体实验设计：秀丽隐杆线虫成虫在存在/不存在bir-2基因敲降、存在/不存在粪肠球菌暴露条件下的全动物mRNA表达谱。