Novel Approaches in Long-Read RNA Sequencing
Increasing fidelity of long-read RNA sequencing with novel template preparation chemistries
High-throughput and single-cell sequencing of coding RNA is hampered by the abundance of ribosomal RNA (rRNA) in cellular RNA preparations. Self-priming of rRNA generates primer-independent, nonspecific background products that interfere with sequencing results. The typical methods for overcoming this problem are removing ribosomal RNA from the sample or enriching for polyA-containing mRNA, both of which require extra processing steps that can introduce bias in the sample.
Dr. Torbett's group is developing strategies and workflows for increasing RNA-seq efficiency. One of their innovations reduces processing steps before sequencing with addition of the CASPR reagent (for chemical ablation of self-priming RNA). CASPR acts at the 3' ends of RNA templates, preventing rRNA self-priming. The Torbett group showed that CASPR is comparable to polyA+ selection for producing samples for RNA-seq and yields comprehensive gene body coverage for protein-coding transcripts. Compared to polyA+ selection, CASPR treatment of RNA increased the specificity and sensitivity of reverse transcriptase as well as the number and length of sequence reads in tests with two leading reverse transcriptases. CASPR is specific to RNA so it does not interfere with any subsequent reactions.
CASPR is a key component of a strategy for quantitative, full-length RNA-seq by nanopore technology that includes hardware and software developed in the Torbett lab. In proof-of-concept studies, the group showed that a sequencing pipeline that includes CASPR, highly processive reverse transcriptases, and oligo-dT priming obtains full-length reads for simultaneous profiling of HIV virus and its host cells without the need of bias-laden PCR amplification. Recent development work in the Torbett Lab has also shown CASPR can be leveraged for preparation of templates for Direct RNA Sequencing, a strategy that allows sequencing of RNA without prior conversion to cDNA, thus preserving epitranscriptomic information such as RNA modifications.
In addition to improving and simplifying the production of cDNA and preparation of templates for Direct RNA Sequencing, CASPR has specific uses in studying transcriptomes, splicing, viral infection and replication, and other applications that use bulk RNA inputs. For applications that require single-cell resolution, the Torbett lab is currently doing proof-of-concept work to demonstrate use of CASPR for single-cell RNA-seq.
Dr. Torbett is interested in partnerships to improve RNA-seq by developing CASPR, for example as a kit addition, along with his other innovations for long-read sequencing.
Stage of Development
- Preclinical in vitro
- Licensing agreement
- Collaborative research and development
- Sponsored research agreement
- Consultation agreement
- Gallardo CM, Nguyen AT, Olson S…Torbett BE. A quantitative full-length RNA-seq strategy for the simultaneous profiling of poly-adenylated HIV and host cell transcripts from unamplified cDNA. In preparation.
- Gallardo CM, Wang S, Montiel-Garcia DJ…Torbett BE. MrHAMER yields highly accurate single molecule viral sequences enabling analysis of intra-host evolution. Nucleic Acids Research (doi: 10.1093/nar/gkab231).