Long-read genotyping with SLANG (Simple Long-read loci Assembly of Nanopore data for Genotyping)

Premise Most phylogenomic library preparation methods and bioinformatic analysis tools in restriction site-associated DNA sequencing (RADseq)/genotyping-by-sequencing (GBS) studies are designed for use with Illumina data. The lack of alternative bioinformatic pipelines hinders the exploration of long-read multi-locus data from other sequencing platforms. The Simple Long-read loci Assembly of Nanopore data for Genotyping (SLANG) pipeline enables locus assembly, orthology estimation, and single-nucleotide polymorphism (SNP) calling using Nanopore-sequenced multi-locus data. Methods and Results Two test libraries (Leucanthemum spp., Senecio spp.; Compositae) were prepared using an amplified fragment length polymorphism (AFLP)-based method to reduce genome complexity, then Nanopore-sequenced, and analyzed with SLANG. We identified 704 and 448 orthologous loci with 12,368 and 10,048 SNPs, respectively. The constructed phylogenetic networks were identical to a GBS network produced using Leucanthemum Illumina data and were consistent with Senecio species circumscriptions based on morphology. Conclusions SLANG identifies orthologous loci and extracts SNPs from long-read multi-locus Nanopore data for phylogenetic inference, population genetics, or phylogeographical studies. Combined with an AFLP-based library preparation, SLANG provides an easily scalable, cost-effective, and affordable alternative to Illumina-based RADseq/GBS procedures.