9.3 Microbial metatranscriptomics

Sequencing library preparation for microbial metatranscriptomics faces the same challenges as host genomics, but the fact that prokaryotic mRNA have no poly-A tails makes it impossible to apply oligo(dT)-based rRNA depletion strategies. There are three other alternatives through which prokaryotic rRNA can be depleted. These three strategies require designing oligos, probes or guides whose sequences complement the DNA sequences that should be removed. Most commercial kits contain probes designed to remove rRNA sequences of the most commonly employed animal hosts (Human/Mouse/Rat), as well as bacteria, but custom probes targeting any genes could be employed. The first two methods shown below are implemented before library-preparation, thus independent reactions must be ran for each sample. The last strategy is implemented after library preparation, which enables multiple indexed libraries to be pooled, and thus performing a single reaction per pool.

Capture-based rRNA depletion

This method relies on capture rRNA with complimentary oligos that are coupled to paramagnetic beads. Unwanted transcripts get bound to beads, which can then be retained using a magnet, while the non-hybridasing transcripts remain in the elution.

RNAse-based rRNA depletion

A more recent technological upgrade to capture-based rRNA depletion is to, instead of using paramagnetic beads, degrade RNA:DNA hybrids using RNase H [34].

CRISPR/Cas9-based rRNA depletion

The newest method of all three relies on the DNA claveage capacity of the Cas9 enzyme [35]. In this method, custom-designed guides are used for the Cas9 enzyme to cleveage unwanted sequences. This strategy is applied once libraries are prepared, and before the final PCR amplification is conducted. When the targetted molecules are cleaveged, they lack one of the two adaptors, and therefore they are not amplified, resulting in a considerable depletion compared to the rest of the library.

List of available protocols

Name	Strategy	Author/owner	Protocol/Article
Custom capture-based depletion	Capture-based	Open Source	Article [36]
Legacy Ribo-Zero	Capture-based	Illumina
Custom RNAse-based depletion	RNAse-based	Open Source	Article [34]
Ribo-Zero Plus	RNAse-based	Illumina
NEBNext® rRNA Depletion Kit	RNAse-based	NEB
DASH	Cas9-based	Open Source	Article [37]

Contents of this section were created by Antton Alberdi.

References

34. Huang Y, Sheth RU, Kaufman A, Wang HH. Scalable and cost-effective ribonuclease-based rRNA depletion for transcriptomics. Nucleic Acids Res. 2020;48:e20.

35. Gu W, Crawford ED, O’Donovan BD, Wilson MR, Chow ED, Retallack H, et al. Depletion of abundant sequences by hybridization (DASH): Using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications. Genome Biol. 2016;17:41.

36. Kraus AJ, Brink BG, Siegel TN. Efficient and specific oligo-based depletion of rRNA. Sci Rep. 2019;9:12281.

37. Prezza G, Heckel T, Dietrich S, Homberger C, Westermann AJ, Vogel J. Improved bacterial RNA-seq by Cas9-based depletion of ribosomal RNA reads. RNA. 2020;26:1069–78.