The (meta)transcriptome constitutes the genes expressed by microbes within any given sample. The study of this through the sequencing and analysis of RNA can give insight into how the microbiome or isolated bacteria behaves under conditions of interest, gaining a deeper understanding of the gene expression and associated functional outcomes.
Given the flexibility afforded by many sample collection kits today, microbiome scientists are becoming increasingly interested in the functional activity of the microbiome under different conditions, disease states, time points and other cohort variations to identify links between microbial functions and a phenotype of interest.
Through optimized RNA-based workflows for a range of different sample types, CosmosID offers (meta)transcriptomic services for both complex samples and microbial isolates for a range of study applications.
The advantages are:
CosmosID’s CLIA-certified, GCP compliant facility accepts samples globally. Regardless of the research area, the lab procedures followed are under the same level of scrutiny, ensuring quality and reproducability for all study types. Upon arrival, your samples will first be barcoded & accessioned into the CosmosID LIMS system for sample tracking and effective project management.
Samples will undergo a validated RNA extraction protocol optimized for shotgun sequencing and customised per sample type. The extracted RNA will undergo an rRNA depletion, where then double-stranded cDNA is then synthesised, ready for sequencing!
Once ds-cDNA QC metrics are established for received or generated cDNA, these samples then undergo library preparation & QC prior to sequencing on an appropriate Illumina instrumentation, depending on the necessary sequencing depth & client preference, if applicable.
Very similarly to metagenomic functional profiling, at CosmosID we query databases such as UniProt to identify functional proteins that have been well annotated. From there, these hits are annotated with MetaCyc reactions (metabolic enzymes) to reconstruct and quantify complete pathways in the microbial community sample. Furthermore, the UniProt gene families are mapped to GO terms. Lastly, to facilitate comparisons across multiple samples with different sequencing depths, the abundance values are normalized using Total-sum scaling (TSS) normalization to produce “Copies per million” (analogous to TPMs in RNA-Seq) units.
Finally, you will have a laundry list of expressed genes within your samples that can be used in downstream analyses such as differential abundance, beta diversity and more! For interest in downstream analysis and statistics or cutom bioinformatics, please enquire below.
For a more comprehensive view on functional potential, metatranscriptomics is a robust method of identifying candidate genes and associating these with traits of interest.
Common applications include but are not limited to:
CosmosID has the ability to provide a wide range of analyses based on RNA workflows including functional profiling to monitor gene expression in complex samples, detection of RNA viruses or looking at activity based on present taxa.
Given the stability of RNA, CosmosID recommends using a microbiome preservation medium for collection and shipping of samples prior to -80C storage. Providing collection is deemed appropriate and likely to be successful, we can accept raw samples, RNA, ds-cDNA and sequenced data.
One of the biggest advantages of metatranscriptomic data is that it is possible to identify genes that are expressed as opposed to those that are simply present and have the potential to be active. As such, by correlating these genes with targeted or untargeted metabolomic profiles, one can start to investigate which metabolites are produced by the microbiome vs the host, and which of those can be linked to a phenotype of interest. From here, mechanism-of-action can start to be investigated more robustly than with either method in isolation.