TI Food and Nutrition (TIFN), P,O, Box 557, 6700 AN, Wageningen, The Netherlands.
BMC Genomics. 2013 Aug 2;14:530. doi: 10.1186/1471-2164-14-530.
Next generation sequencing (NGS) technologies can be applied in complex microbial ecosystems for metatranscriptome analysis by employing direct cDNA sequencing, which is known as RNA sequencing (RNA-seq). RNA-seq generates large datasets of great complexity, the comprehensive interpretation of which requires a reliable bioinformatic pipeline. In this study, we focus on the development of such a metatranscriptome pipeline, which we validate using Illumina RNA-seq datasets derived from the small intestine microbiota of two individuals with an ileostomy.
The metatranscriptome pipeline developed here enabled effective removal of rRNA derived sequences, followed by confident assignment of the predicted function and taxonomic origin of the mRNA reads. Phylogenetic analysis of the small intestine metatranscriptome datasets revealed a strong similarity with the community composition profiles obtained from 16S rDNA and rRNA pyrosequencing, indicating considerable congruency between community composition (rDNA), and the taxonomic distribution of overall (rRNA) and specific (mRNA) activity among its microbial members. Reproducibility of the metatranscriptome sequencing approach was established by independent duplicate experiments. In addition, comparison of metatranscriptome analysis employing single- or paired-end sequencing methods indicated that the latter approach does not provide improved functional or phylogenetic insights. Metatranscriptome functional-mapping allowed the analysis of global, and genus specific activity of the microbiota, and illustrated the potential of these approaches to unravel syntrophic interactions in microbial ecosystems.
A reliable pipeline for metatransciptome data analysis was developed and evaluated using RNA-seq datasets obtained for the human small intestine microbiota. The set-up of the pipeline is very generic and can be applied for (bacterial) metatranscriptome analysis in any chosen niche.
下一代测序（NGS）技术可应用于复杂微生物生态系统，通过直接 cDNA 测序进行宏转录组分析，这被称为 RNA 测序（RNA-seq）。RNA-seq 产生了大量非常复杂的数据集，要全面解释这些数据集，需要一个可靠的生物信息学管道。在本研究中，我们专注于开发这种宏转录组管道，并使用来自两个人造口小肠微生物群的 Illumina RNA-seq 数据集对其进行验证。
这里开发的宏转录组管道能够有效地去除 rRNA 衍生序列，然后自信地分配预测的功能和 mRNA reads 的分类起源。小肠宏转录组数据集的系统发育分析与从 16S rDNA 和 rRNA 焦磷酸测序获得的群落组成图谱具有很强的相似性，这表明群落组成（rDNA）与微生物成员的整体（rRNA）和特定（mRNA）活性的分类分布之间存在相当大的一致性。通过独立的重复实验建立了宏转录组测序方法的可重复性。此外，比较使用单端或配对末端测序方法进行的宏转录组分析表明，后者方法并不能提供改进的功能或系统发育见解。宏转录组功能映射允许分析微生物群落的全局和属特异性活性，并说明了这些方法在揭示微生物生态系统中的共生相互作用方面的潜力。
使用从人小肠微生物群获得的 RNA-seq 数据集开发并评估了一种可靠的宏转录组数据分析管道。该管道的设置非常通用，可以应用于任何选定生态位的（细菌）宏转录组分析。
