A method for filtering abnormal modified base calling in Oxford Nanopore Technologies sequencing

Abstract

Backgroundgene synthesis sequencing using the long-read Oxford Nanopore Technologies (ONT) provides a cost-effective option for gene synthesis quality control. Despite the advantage of using long reads, however, accurate base calling is influenced by modified bases. ResultsWe introduce a method for filtering abnormal modified base calling in Oxford Nanopore Technologies sequencing. This method is based on the mapping results and perform an exact binomial test on the proportion of single base forward and reverse chain depth to determine the presence of abnormal modified base calling. Based on Sanger sequencing results, this method detected 96.79% of effective abnormal modified base calling, and the accuracy of gene synthesis sequencing increased from 99.40% before calibration to 99.98% after calibration, significantly reducing the proportion of requiring secondary gene synthesis sequencing. ConclusionThis method can accurately filter abnormally modified base calling sites, reduce the proportion of using Sanger for secondary sequencing, thereby saving costs, and improving efficiency.