核糖体谱分析是一种新兴的实验技术,通过对核糖体中进行翻译的短mRNA片段进行测序来测量蛋白质合成。在基因组范围内应用,这是一个强大的工具来描述感兴趣的细胞群体中的整体蛋白质合成。此类信息可用于生物标志物发现和治疗响应基因的检测。然而,核糖体谱分析数据的分析需要仔细的预处理,以减少伪影的影响和专门的统计方法,用于可视化和建模高维离散读取计数数据。在这里,我们介绍核糖体剖面分析报告(RP-REP),一种新的开源云软件,允许用户在托管在AWS上或用户自己的UbuntuLinux服务器上的预配置AmazonVirtualMachineImage(AMI)上执行从基因水平到末端的核糖体分析和RNA-Seq分析。该软件与本地存储的FASTQ文件一起工作,在AWSS3上,或在序列读取存档(SRA)上。RP-REP自动执行一系列可定制的步骤,包括污染物RNA的过滤,真正的核糖体足迹的富集,参考比对和基因翻译定量,基因体覆盖,CRAM压缩,参考校准QC,数据规范化,多变量数据可视化,差异翻译基因的鉴定,和热图的生成,共翻译基因簇,丰富的途径,和其他自定义可视化。RP-REP提供了对比RNA-SEQ和核糖体分析结果的功能,并计算每个基因的翻译效率。该软件输出PDF报告和可发布的表格和图形文件。作为一个用例,我们为登革热病毒研究提供RP-REP结果,该研究测试了人Huh7细胞感染前和6小时的胞质溶胶和内质网细胞部分,12h,24h,感染后40小时。案例研究结果,Ubuntu安装脚本,和最新的RP-REP源代码可以在GitHub上访问。云就绪AMI可在AWS上获得(AMIID:RPREPRSEQREP(核糖体分析和RNA-Seq报告)v2.1(ami-00b92f52d763145d3))。
Ribosomal profiling is an emerging experimental technology to measure protein synthesis by sequencing short mRNA fragments undergoing translation in ribosomes. Applied on the genome wide scale, this is a powerful tool to profile global protein synthesis within cell populations of interest. Such information can be utilized for biomarker discovery and detection of treatment-responsive genes. However, analysis of ribosomal profiling data requires careful preprocessing to reduce the impact of artifacts and dedicated statistical methods for visualizing and modeling the high-dimensional discrete read count data. Here we present Ribosomal Profiling
Reports (RP-REP), a new open-source cloud-enabled software that allows users to execute start-to-end gene-level ribosomal profiling and RNA-Seq analysis on a pre-configured Amazon Virtual Machine Image (AMI) hosted on AWS or on the user\'s own Ubuntu Linux server. The software works with FASTQ files stored locally, on AWS S3, or at the Sequence Read Archive (SRA). RP-REP automatically executes a series of customizable steps including filtering of contaminant RNA, enrichment of true ribosomal footprints, reference alignment and gene translation quantification, gene body coverage, CRAM compression, reference alignment QC, data normalization, multivariate data visualization, identification of differentially translated genes, and generation of heatmaps, co-translated gene clusters, enriched pathways, and other custom visualizations. RP-REP provides functionality to contrast RNA-SEQ and ribosomal profiling results, and calculates translational efficiency per gene. The software outputs a PDF report and publication-ready table and figure files. As a use
case, we provide RP-REP results for a dengue virus study that tested cytosol and endoplasmic reticulum cellular fractions of human Huh7 cells pre-infection and at 6 h, 12 h, 24 h, and 40 h post-infection.
Case study results, Ubuntu installation scripts, and the most recent RP-REP source code are accessible at GitHub. The cloud-ready AMI is available at AWS (AMI ID: RPREP RSEQREP (Ribosome Profiling and RNA-Seq
Reports) v2.1 (ami-00b92f52d763145d3)).