Full-length PacBio Amplicon Sequencing to Unveil RNA Editing Sites

RNA editing enriches post-transcriptional sequence changes. Currently detecting RNA editing sites is mostly based on the Sanger sequencing platform and second-generation sequencing. However, detection with Sanger sequencing is limited by the disturbing background peaks using the direct sequencing method and the clone number using the clone sequencing method, while second-generation sequencing detection is constrained by its short read. We aimed to design a pipeline that can accurately detect RNA editing sites for full-length long-read amplicons to meet the requirement when focusing on a few specific genes of interest. We developed a novel high-throughput RNA editing sites detection pipeline based on the PacBio circular consensus sequences sequencing which is accurate with high-throughput and long-read coverage. We tested the pipeline on cytosolic malate dehydrogenase in the hard-shelled mussel Mytilus coruscus and further validated it using direct Sanger sequencing. Data generated from the PacBio circular consensus sequences (CCS) amplicons in three mussels were first filtered by quality and then selected by open reading frame. After filtering, 225-2047 sequences of the three mussels, respectively, were used to identify RNA editing sites. With corresponding genomic DNA sequences, we extracted 227-799 candidate RNA editing sites excluding heterozygous sites. We further figured out 7-11 final RESs using a new error model specially designed for RNA editing site detection. The resulting RNA editing sites all agree with the validation using the Sanger sequencing. We report a near-zero error rate method in identifying RNA editing sites of long-read amplicons with the use of PacBio CCS sequencing.