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!
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:
By leveraging metatranscriptomics, we can delve into the complexities of the microbial community by enabling us to understand the expressed functional potential of the microbiome.
Offering a different perspective into microbiome sciences, metatranscriptomics helps facilitate more focused research into the microbiome’s functional capacity within samples, When paired with CosmosID’s state-of-the-art software platforms, which enhances the accessibility and usability of this approach, researchers generate a wealth of data around the gene expressions of organisms.
Metatranscriptomics is a powerful solution that can elucidate the connections within microbial functions, diseases and medical diagnosis, treatment production, environmental conditions, and different physiological states through analysis of the community RNA within a sample. It can help us understand the mechanisms behind observed gene expressions, and guide us to new discoveries in the field of microbiome..
Metatranscriptomics is employed for a wide array of applications aimed at understanding complex microbial communities and their influence on various domains.
In health-related research, this method has been instrumental in uncovering the links between microbial gene expression profiles and human health, supporting research in conditions such as inflammatory bowel disease and Alzheimer’s..
With its ability to provide functional annotations of differentially expressed genes, metatranscriptomics is also widely used in pharmaceutical and nutritional studies for clinical sample analysis.
Beyond human health, the agricultural and environmental sectors harness this approach for research into crop health and soil microbiome respectively. Additionally, metatranscriptomics is utilized in the exploration and characterization of probiotics and LBPs (Live Biotherapeutic Products), bringing a new dimension to the field of therapeutic research.
The metatranscriptomics method begins with the collection of samples, which are then subjected to RNA extraction and rRNA (Ribosomal RNA) depletion. The extracted RNA is converted into cDNA, which is then prepared for sequencing.
High-throughput sequencing technologies, particularly Illumina instrumentation, are then employed to determine the gene expression profiles of the microbial communities in the sample.
The generated sequences are then mapped onto a reference genome or a reference database to identify the active genes and their corresponding functional annotations.
Subsequent data analysis involves querying reference databases such as UniProt to identify functional proteins, which are then annotated with metabolic enzymes for pathway reconstruction. Statistical analysis of the abundant values allows for the functional analysis of the community, with differentially expressed genes providing insights into active metabolic pathways.
This comprehensive multi-step approach allows for an in-depth understanding of not just the microbiome’s composition, but also its functional dynamics, thereby providing a host and microbial RNA-based snapshot of the microbial activity in various environments.
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.
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.