Chemical capping improves template switching and enhances sequencing of small RNAs

Abstract

Template-switching reverse transcription is widely used in RNA sequencing for low-input and low-quality samples, including RNA from single cells or formalin-fixed paraffin-embedded (FFPE) tissues. Previously, we identified the native eukaryotic mRNA 5′ cap as a key structural element for enhancing template switching efficiency. Here, we introduce CapTS-seq, a new strategy for sequencing small RNAs that combines chemical capping and template switching. We probed a variety of non-native synthetic cap structures and found that an unmethylated guanosine triphosphate cap led to the lowest bias and highest efficiency for template switching. Through cross-examination of different nucleotides at the cap position, our data provided unequivocal evidence that the 5′ cap acts as a template for the first nucleotide in reverse transcriptase-mediated post-templated addition to the emerging cDNA—a key feature to propel template switching. We deployed CapTS-seq for sequencing synthetic miRNAs, human total brain and liver FFPE RNA, and demonstrated that it consistently improves library quality for miRNAs in comparison with a gold standard template switching-based small RNA-seq kit.

Construction of multiplexed transcriptome NGS libraries of microdissected tissue samples based on combinational DNA barcode microbeads

“…[ 32 ] However, the lack of barcode‐based primers limits its application to high‐throughput, multiplexed cellular studies, particularly with increasingly advanced single‐cell resolution sampling techniques. In addition to Smart‐3SEQ, there are many other studies targeting FFPE samples for transcriptome analysis, such as high‐throughput single‐nucleus transcriptome analysis of FFPE samples using random primer, [ 42 ] the study of small RNA by combining chemical capping and template switching, [ 43 ] and tumor investigation by exome sequencing technology. [ 44 ] However, these techniques often involve tedious paraffin removal, hydration, and other operations.…”

To make a short story long: simultaneous short and long RNA profiling on Nanopore devices

“…This is where the RT switches templates and adds non-templated nucleotides. Previous work that examined the template switching junction has shown both sequence dependent bias (63)(64)(65)(66) and variable numbers of non-template nucleotides added (64). Therefore, neither the length, nor the composition of the junction can be taken as granted, and thus we opted to retain this feature as part of the insert sequence during text segmentation.…”

A cap 0-dependent mRNA capture method to analyze the yeast transcriptome

“…Unlike eIF4E, IFIT1 has no dependence on N7-methylation, which could facilitate mRNA capture in suboptimal or degraded samples (in which N7-methyl group may be lost or inefficiently introduced). This property of IFIT1 could also be advantageous in studies using synthetic RNA ( 22 ). This includes applications in which the unmethylated cap is preferred, such as RNA sequencing pipelines using template-switching reverse transcription, in which GpppRNA confers improved efficiency and less bias than m7GpppRNA ( 22 ).…”

“…This property of IFIT1 could also be advantageous in studies using synthetic RNA ( 22 ). This includes applications in which the unmethylated cap is preferred, such as RNA sequencing pipelines using template-switching reverse transcription, in which GpppRNA confers improved efficiency and less bias than m7GpppRNA ( 22 ). Therefore, IFIT1 offers complementary properties to those of eIF4E: whereas IFIT1 is not expected to pull-down the cap 1 or cap 2 mRNA of vertebrates (to which our method would thus not apply), it is suitable for enrichment of mRNA lacking methyl groups at the 2′O of the first two nucleotides and facultatively the N7 position in the cap.…”

“…Importantly, eIF4E(K119A) or yeast cap-binding proteins failed to enrich yeast mRNA, leaving IFIT1 as the only viable tool for 5′ end-dependent RNA capture in this species that can effectively replace eIF4E in such applications. Due to the high specificity of IFIT1 for cap 0, we believe that our method could be particularly useful for transcriptomic analysis in model and non-model lower eukaryotes or those relying on template-switching reverse transcription ( 22 ). We show that an IFIT-based approach can serve as a robust substitute method to commercially available approaches.…”