Exploring the functional activity of microbial communities
profiling of community-wide gene expression (RNA-seq)
While Metagenomics tells us which microbes are present and what genomic potential they have, Metatranscriptomics tells us about their activity: the genes that are highest expressed in a specific mircobial environement. Thus, Metatranscriptomics is the study of the function and activity of the complete set of transcripts (RNA-seq) from environmental samples.
Gene activity diversity
How many different genes are expressed in a microbial community across all species? How is the functional and pathway diversity?
Key genes and pathways of highest activity
Which are the highest expressed genes in a specific environmental condition? What is the most important functionality (highly activated pathway) needed in an environment?
Gene expression is log-like distributed: Top 100 genes of highest expression can cover up to 30% of all transcripts. Even a single gene can cover up to 10%. Thus, a very high sequencing depth is required to see also lower expressed genes.
Gene expression comparison
Which genes show the highest change in expression levels between different conditions (biomarker detection)?
Before comparison, expression levels can be normalized by RPKM to correct for differences in sequencing depth and gene length.
Kallisto - RNA-seq transcript abundance estimation
Kraken - assigns taxonomic labels to short sequencing reads
HUMAnN2 - community-wide gene transcription and pathway analysis
requires pairs of DNA and RNA sequencing of the same biological sample (to get corrected RNA transcription levels, normalized by the sample's DNA bacteria abundance)
PanPhlAn - strain-specific transcriptomics analysis
requires pairs of DNA and RNA sequencing of the same biological sample