<br/>Template switching technologies are important to the application of several RNA sequencing methodologies, particularly for reduced bias relative to traditional ligation-based approaches. Single-cell RNA sequencing (scRNA-seq) takes advantage of the low input amounts of RNA provided by template switching and has led to key insights into gene expression of individual cells. In our previous study, we identified the 5’ cap structure as an integral component to reducing bias from template switching reverse transcription and found further evidence that nontemplated addition and template switching are concurrent and competing processes. To expand upon our previous study, we here present a chemical capping method that can be applied to small RNA sequencing. First, we synthesized a collection of diverse chemically capped 25mer RNAs and studied their template switching efficiency and nontemplated additions using an MMLV-based reverse transcriptase. We found that unmethylated Gppp caps provide the lowest bias and highest template switching efficiency for all 5’ end nucleotides, and we showed further evidence that the 5’ cap indeed acts as a template for nontemplated additions by the reverse transcriptase. We then applied the Gppp cap to a library preparation workflow (called CapTS-seq), and sequenced synthetic miRNAs, total RNA, and FFPE RNA, finding that consistently, the chemical capping method in conjunction with template switching improves sequencing bias and library quality in comparison to the commercially available SMARTer smRNA seq kit. Finally, we showed that with adjusted sequencing parameters, we detected fragmented mRNA in FFPE total RNA, highlighting the potential for the chemical capping workflow in the detection of multiple classes of RNA.

Improving sequencing biases in RNA detection through chemical capping and template switching

ACS Spring Meeting 2021

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.