Alternative splicing is a regulated process by which eukaryotic genes may produce diverse biological products. Defects in the process typically affect cellular function and can lead to disease. Next-generation sequencing (NGS) technologies have been developed to detect alternative splicing events; however, the alternative splicing events detected by standard RNA-Seq may or may not be derived from full-length RNA. The SMARTer method provides full-length double-strand cDNA synthesis, and the resulting gene expression patterns correlate strongly with standard RNA-Seq. However, it also yields non-specific genomic DNA amplification. We improved the SMARTer method by employing a target-capture full-length double-strand cDNA sequencing method. High-fidelity, full-length cDNA is generated by the SMARTer method, followed by target-specific capture with exon probes. The expression pattern observed with this SMARTer Capture method was highly correlated with the results of the original SMARTer method. The number and accuracy of the detected splicing events were increased by eliminating non-specific genomic DNA amplification by the SMARTer Capture. Compared to the original SMARTer method, the SMARTer Capture provided 4-fold greater detection of alternative splicing events at the same read number, and it took less than 1/100 of read number to detect the same number of splicing events. The percent splicing in index (PSI) of the SMARTer Capture is highly correlated with the PSI of the SMARTer. These results indicate that the SMARTer Capture represents an improvement of the SMARTer method to accurately characterize alternative splicing repertories in targeted genes without biases.
- Alternative splicing
- RNA-Sequencing (RNA-Seq)
- full-length double-strand cDNA
- next-generation sequencing
- splicing variant