Next-generation sequencing (NGS) has revolutionized genomics research, allowing scientists to explore the intricacies of DNA and RNA at an unprecedented scale. However, the quality of NGS data heavily relies on the removal of unwanted sequences that can interfere with accurate analysis. These unwanted sequences include abundant ribosomal RNA (rRNA), host DNA, or contaminants. To address this challenge, researchers have developed various depletion technologies. In this blog, we delve into the advantages and limitations of existing depletion approaches, including hybridization, RNase H, CRISPR, and kinetic folding.

Hybridization-based depletion

Hybridization relies on complementary base pairing between target sequences and specific probes. For rRNA depletion, researchers design probes complementary to rRNA sequences. These probes can be immobilized on beads or magnetic particles.

Advantages:

• Specificity: Hybridization probes can be designed to target specific RNA or DNA sequences. This specificity ensures that only the intended sequences are removed.
• Customizability: Researchers can tailor probes to different organisms, RNA species, or even specific genes.
• Ease of use: Hybridization-based methods are relatively straightforward and widely applicable.

Limitations:

• Probe design challenges: Designing effective probes requires knowledge of the target sequences. For non-model organisms or poorly characterized RNA species, probe design can be challenging.
• Off-target effects: Probes may hybridize to unintended sequences, leading to incomplete depletion or false positives.

RNase H-mediated depletion

RNase H specifically cleaves RNA in RNA-DNA hybrids. Synthetic DNA oligonucleotides complementary to rRNA are used. When hybridized to rRNA, RNase H degrades the RNA strand.

Advantages:

• Specific cleavage: RNase H specifically targets RNA-DNA hybrids, ensuring precise removal of unwanted RNA.
• Minimal reagents: RNase H-mediated depletion requires only the enzyme and complementary DNA oligonucleotides.
• Cost-effective: The simplicity of this method makes it cost-effective.

Limitations:

• RNA-DNA hybrids: RNase H only acts on RNA-DNA hybrids. If the target RNA is single-stranded, this method is ineffective.
• Sequence-specificity: The efficiency of RNase H depends on the complementarity of the DNA oligos to the target RNA.

CRISPR/Cas9-based depletion

CRISPR/Cas9 is known for genome editing but can also be used for depletion of double stranded DNA molecules. Guide RNAs (gRNAs) are designed to target DNA sequences. Cas9 protein cleaves the libraries containing the target sequence, which are then easily removed by size selection.

Advantages:

• Versatility: CRISPR/Cas9 can be customized for various RNA or DNA targets beyond rRNA.
• High specificity: Guide RNAs (gRNAs) precisely target the desired sequences.
• Flexibility: Researchers can adapt this method to different organisms and experimental needs.

Limitations:

• Design complexity: Designing effective gRNAs requires bioinformatics expertise.

Kinetic folding-based depletion

Uses the repetitive properties of ribosomal and long non-coding RNA sequences, at an optimal temperature, to inhibit reverse transcription.

Advantages:

• No additional reagents: Kinetic folding relies on the inherent properties of RNA molecules.
• Simplicity: No need for specific probes or enzymes.

Limitations:

• RNA secondary structure: Kinetic folding depends on the differential folding rates of RNA species. Complex secondary structures can affect the efficiency.
• Fragment size: This approach has limitations based on fragment size (i.e. FFPE) and tends to lose high GC rich transcripts as well due to the suboptimal temperature for reverse transcription
• Limited targeting: This method primarily focuses on rRNA. Customization for other targets is challenging. It does not work on double-stranded DNA

Conclusion

Depletion technologies are essential for enhancing the accuracy of NGS experiments. Researchers must choose the most suitable approach based on their specific goals and sample characteristics. As NGS continues to evolve, these depletion strategies will play a crucial role in unlocking the secrets of genomics.

For research use only. Not for use in diagnostic procedures.

References

• Wahl, A., Huptas, C. & Neuhaus, K. (2022) Comparison of rRNA depletion methods for efficient bacterial mRNA sequencing. Sci Rep 12, 5765
• Jang, J.S., Berg, B., Holicky, E. et al. (2020). Comparative evaluation for the globin gene depletion methods for mRNA sequencing using the whole blood-derived total RNAs. BMC Genomics 21, 890.
• Zhao, S., Zhang, B. (2015). A comprehensive evaluation of ensembl, RefSeq, and UCSC annotations in the context of RNA-seq read mapping and gene quantification. BMC Genomics, 16, 1-11. DOI: 10.1186/s12864-015-1318-0
• Levin, J. Z., Yassour, M., Adiconis, X., Nusbaum, C., Thompson, D. A., Friedman, N., Gnirke, A., & Regev, A. (2010). Comprehensive comparative analysis of strand-specific RNA sequencing methods. Nature Methods, 7(9), 709–715. DOI: 10.1038/nmeth.1491.