Validating Oxford Nanopore Technologies Amplicon Sequencing as an Alternative to Sanger Sequencing for Generating Plant Molecular Markers

Molecular phylogenetic markers used to be ubiquitously obtained by Sanger sequencing, while recently, next generation sequencing (NGS) approaches have become popular alternatives. In this study, a pipeline utilizing Oxford Nanopore Technologies (ONT) amplicon sequencing (amplicon‐seq) was examined as an alternative to Sanger sequencing, with nine loci [11 polymerase chain reaction (PCR) products], in Aeschynanthus (Gesneriaceae). Pooled PCR products for each species were sequenced on an ONT MinION system. For scalability and ease of consensus sequence acquisition, a python ‘snakemake’ bioinformatic pipeline, ‘petithebi’ (small snake), was designed. To assess the required minimum coverage, we simulated various coverage levels by using different numbers of input reads and found no nucleotide polymorphisms, but a few indels at polymononucleotide sites at low coverage. When comparing ONT amplicon‐seq with Sanger sequencing, only two out of 81 sequences differed, involving one nucleotide change and one indel, demonstrating a very high reproducibility between the two methods. Thus, ONT amplicon‐seq is a suitable alternative for Sanger sequencing to obtain molecular markers, also allowing Sanger legacy data to be combined with NGS‐derived ONT amplicon‐seq data. The ‘ petithebi’ pipeline is also a useful tool to ease the transition from Sanger sequencing to NGS, where a high demand of bioinformatic skill is often a barrier.