Enabling Functional Profiling of Human Metatranscriptomes Using RiboZero™ Plus Microbiome

Human microbiomes are composed of highly complex mixtures of microbes that can dynamically adjust their gene expression in response to environment, diet, exercise or even disease states. Indeed, the content and condition of the human gut microbiome can have profound influence on the health of the host. Profiling human microbiomes is particularly hampered by the highly abundant rRNA that dominates the samples and can represent ~98-99% of sequencing reads.

Removal of the rRNA by any depletion method is a challenging endeavor since the samples contain hundreds of different bacterial, archeal and fungal, greatly increasing the diversity of rRNA sequences encountered. Designing rRNA depletion probes targeting all the taxa present in human gut, for example, will result in far too many probes to be of practical use, so a more directed approach is needed. We have created a rational probe design strategy that is not based upon taxonomy, but instead relies on mining the most abundant sequences found in the raw fastq files from sequenced samples of multiple donors.

This approach, together with strategic filtering based upon coverage and pairwise alignments to reduce redundancy, results in a far more manageable number of probes that can effectively deplete rRNA from several distinct human microbiome types. These probes are used in an enzymatic-based depletion method and is commercialized as the RiboZero Plus Microbiome kit. Effective removal of the rRNA results in far more reads being assigned to the functional metatranscriptomes in these sample types, thus enabling sensitive profiling of microbial diversity and metabolic activity as well as comparisons between the health or even disease states of the host.

Furthermore, we have also created an informatics tool to aid in the study of metatranscriptomes called the Microbiome Metatranscriptomics App in Illumina’s BaseSpace Sequencing Hub. This app links several tools that filter out the rRNA reads, perform taxonomic identification as well as functional profiling and gene ontology of species present in the microbiome samples.

Scott Kuersten
Sr. Staff Scientist
Illumina, Inc.

Brice Le François, Ph.D.
Senior R&D Manager – Technology
DNA Genotek Inc.