Full-length circRNA sequencing method using low-input RNAs and profiling of circRNAs in MPTP-PD mice on a nanopore platform†

Considering the importance of accurate information of full-length (FL) transcripts in functional analysis, researchers prefer to develop new sequencing methods based on third-generation sequencing (TGS) rather than short-read sequencing. Several FL circRNA sequencing strategies have been developed. However, the current methods are inapplicable to low-biomass samples, since a large amount of total RNAs are acquired for circRNA enrichment before library preparation. In this work, we developed an effective method to detect FL circRNAs from a nanogram level (1–100 ng) of total RNAs based on a nanopore platform. Additionally, prior to the library preparation process, we added a series of 24 nt barcodes for each sample to reduce the cost and operating time. Using this method, we profiled circRNA expression in the striatum, hippocampus and cerebral cortex of a Parkinson's disease (PD) mouse model. Over 6% of reads were effective for FL circRNA identification in most datasets. Notably, a reduction in the RNA initial input resulted in a lower correlation between replicates and the detection efficiency for longer circRNA, but the lowest input (1 ng) was able to detect numerous FL circRNAs. Next, we systematically identified over 263 934 circRNAs in PD and healthy mice using the lower-input FL sequencing method, some of which came from 50.52% of PD-associated genes. Moreover, significant changes were observed in the circRNA expression pattern at an isoform level, and high-confidence protein translation evidence was predicted. Overall, we developed an effective method to characterize FL circRNAs from low-input samples and provide a comprehensive insight into the biological function of circRNAs in PD at an isoform level.