AmpliPiper: A versatile amplicon-seq analysis tool for multilocus DNA barcoding

The advent of third generation sequencing technology has revolutionized parallelized sequencing of DNA fragments of varying lengths, such as PCR amplicons, which provides unprecedented new opportunities for large-scale and diverse DNA barcoding projects that, for example, aim to quantify the accelerating biodiversity crisis. However, the broad-scale application of these new technologies for biodiversity research is often hindered by the demand for advanced bioinformatics skills to carry out quantitative analyses. To facilitate the application of multilocus amplicon sequencing (amplicon-seq) data for biodiversity and integrative taxonomic research questions, we present AmpliPiper, an automated and user-friendly software pipeline which carries out bioinformatics analyses of multilocus amplicon-seq data generated with Oxford Nanopore (ONT) sequencing. AmpliPiper combines analysis methods for DNA barcoding data that include demultiplexing of pooled amplicon-seq data, haplotype-specific consensus sequence reconstruction, species identification based on comparison to the BOLD and GenBank databases, phylogenetic analyses and species delimitation. We demonstrate the applicability and workflow of our approach based on a newly generated dataset of 14 hoverfly (Syrphidae) samples that were amplified and sequenced at four marker genes. We further benchmark our approach with Sanger sequencing and simulated amplicon-seq data which show that DNA barcoding with ONT is both accurate and sensitive to detect even subtle genetic variation.