To assemble or not to resemble -- A validated Comparative Metatranscriptomics Workflow (CoMW)
BackgroundMetatranscriptomics has been used widely for investigation and quantification of microbial communities activity in response to external stimuli. By assessing the genes expressed, metatranscriptomics provide an understanding of the interactions between different major functional guilds and the environment. Here, we present de-novo assembly-based Comparative Metatranscriptomics Workflow (CoMW) implemented in a modular, reproducible structure, significantly improving the annotation and quantification of metatranscriptomes. Metatranscriptomics typically utilize short sequence reads, which can either be directly aligned to external reference databases (\"assembly-free approach\") or first assembled into contigs before alignment (\"assembly-based approach\"). We also compare CoMW (assembly-based implementation) with assembly-free alternative workflow, using simulated and real-world metatranscriptomes from Arctic and Temperate terrestrial environments. We evaluate their accuracy in precision and recall using generic and specialized hierarchical protein databases.\n\nResultsCoMW provided significantly fewer false positives resulting in more precise identification and quantification of functional genes in metatranscriptomes. Using the comprehensive database M5nr, the assembly-based approach identified genes with only 0.6% false positives at thresholds ranging from inclusive to stringent compared to the assembly-free approach yielding up to 15% false positives. Using specialized databases (Carbohydrate Active-enzyme and Nitrogen Cycle), the assembly-based approach identified and quantified genes with 3-5x less false positives. We also evaluated the impact of both approaches on real-world datasets.\n\nConclusionsWe present an open source de-novo assembly-based Comparative Metatranscriptomics Workflow (CoMW). Our benchmarking findings support the argument of assembling short reads into contigs before alignment to a reference database, since this provides higher precision and minimizes false positives.