At Microbiome Insights, we are now pleased to offer metatranscriptomic sequencing services, providing information on gene expression and associated functional outcomes for a microbial community. Our metatranscriptomic sequencing services are backed by cutting-edge high-throughput sequencers, sequencing strategies, and advanced bioinformatics pipelines.
What is metatranscriptomics?
Metatranscriptomics is the study of all RNA molecules (the transcriptome) produced by all the microorganisms present in a particular environment, such as soil, water, or the human gut. High-throughput RNA sequencing can provide insights into the gene expression patterns, and information on metabolic activities and functional roles of different microbial species in an ecosystem. Metatranscriptomics focuses on messenger RNA analysis, though rRNA gene analysis is also possible if interested in targeting only active communities.
As mRNA is short-lived, with a half-life measured in minutes, it represents a precise snapshot of what is happening moment to moment in a sample. Using mRNA to capture transcriptional changes through sequencing is thus a powerful tool to investigate the cellular response to various conditions experienced by microorganisms.
Metatranscriptomics is useful for:
- Gaining insights into gene expression rather than genes alone
- Pinpointing active metabolic pathways
- Identifying mechanisms by which gene expression is occurring
Metatranscriptomics has applications in various fields, including environmental microbiology, biotechnology, and medical research. Samples suitable for metatranscriptomic sequencing include but are not limited to: fecal, saliva, tissue, and vaginal swabs.
Metatransciptomics-as-a-service offers academic, clinical and industry researchers looking for the next breakthrough in microbial characterization to access cutting-edge technology without investing in expensive infrastructure and advanced technical knowledge.
Since mRNA is unstable and short-lived it is very important to select a sampling period and location in which you expect your activity of interest to be occurring. For example, if you are interested in cold resistance genes, you need to sample after a transition to cold conditions. Additionally, special care is needed to stabilize the sample using very low temperatures (e.g., snap freezing to -80C using liquid nitrogen) or stabilization solutions (e.g., RNAlater) to avoid mRNA degradation.
RNA extraction is the most critical step to obtain high-quality RNA for cDNA synthesis and sequencing. RNA extraction is carried out in our CAP-accredited lab with specialized kits, and RNAse-treated instruments and working environments. After extraction, roughly 80% of the total RNA is ribosomal; therefore, rRNA depletion is sometimes used to reduce the rRNA load and increase the messenger RNA (mRNA) content in the mixture. This procedure varies in efficiency and is not recommended in low biomass samples. Another option is RNA/DNA coextraction, which enables paired metagenomics and metatranscriptomics sequencing.
There are two options for RNA library preparation:
- rRNA depletion OR
- poly(A) mRNA selection.
Option 2 is the preferred method (certainly for prokaryotic/bacterial organisms), as it preserves snoRNA as well as rRNA and mRNA.
Following total RNA extraction and purification, including the removal of residual DNA by DNase treatment, mRNA must be depleted to obtain adequate sequencing coverage. QC checks must be carried out after these processes given that RNA is a fragile and transient molecule.
Prior to sequencing, RNA is generally fragmented, size-selected, and converted to double-stranded cDNA using single-strand cDNA conversion and second-strand preparation. This process eliminates strandedness; analysis depending on strand selection (e.g. Anti-sense Adaptors and barcodes) are then ligated to the cDNA fragments during library preparation.
Bioinformatic pipelines in metatranscriptomics are the next part of analyzing the expression profiles of microbial communities in diverse environments. The processing and analysis of metatranscriptomic data will ultimately depend on the questions being addressed in a particular study. A typical pipeline includes several steps, which are outlined below:
Irene Yang, PhD, RN
Assistant Professor at Emory University
Frequenty Asked Questions
What are the sample requirements?
Samples sources including environmental and clinical samples. The recommended RNA Integrity Number (RIN) > 7, with minimum concentration of 1ng/uL of 50 uL in volume (50 ng total amount).
How many reads are needed?
The number of RNA-depleted reads required largely depends on the anticipated complexity of the sample and the objectives of the study. To assess differential expression of pure cultures, approximately 5 million reads should be sufficient. However, to investigate metatranscriptomes of complex samples, more than 50 million reads are required.
How should samples be stored?
Raw samples should be stored in -70 degrees or with dry ice as quickly as possible, with RNA storage buffers if possible [we recommend RNAlater solution or DNA/RNA Shield].
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