PURC Provides Improved Sequence Inference for Polyploid Phylogenetics and Other Manifestations of the Multiple-Copy Problem

Abstract

Inferring the true biological sequences from amplicon mixtures remains a difficult bioinformatics problem. The traditional approach is to cluster sequencing reads by similarity thresholds and treat the consensus sequence of each cluster as an “operational taxonomic unit” (OTU). Recently, this approach has been improved by model-based methods that correct PCR and sequencing errors in order to infer “amplicon sequence variants” (ASVs). To date, ASV approaches have been used primarily in metagenomics, but they are also useful for determining homeologs in polyploid organisms. To facilitate the usage of ASV methods among polyploidy researchers, we incorporated ASV inference alongside OTU clustering in PURC v2.0, a major update to PURC (Pipeline for Untangling Reticulate Complexes). In addition, PURC v2.0 features faster demultiplexing than the original version and has been updated to be compatible with Python 3. In this chapter we present results indicating that using the ASV approach is more likely to infer the correct biological sequences in comparison to the earlier OTU-based PURC and describe how to prepare sequencing data, run PURC v2.0 under several different modes, and interpret the output.