Abstract Evolutionary changes in populations of microbes, such as microalgae, cannot be traced using conventional metabarcoding loci as they lack intraspecific resolution. Consequently, selection and competition processes amongst strains of the same species cannot be resolved without elaborate isolation, culturing, and genotyping efforts. Bamboozle, a new bioinformatic tool introduced here, scans a species’ entire genome and identifies allele-rich barcodes that enable direct identification of different strains from a common population, and a single DNA sample, using amplicon sequencing. We demonstrate its usefulness by identifying hypervariable barcoding loci (&lt;500 bp) from genomic data in two microalgal species, the diploid diatom Skeletonema marinoi , and the haploid chlorophyte Chlamydomonas reinhardtii . Across the genomes, only 26 loci capable of resolving all available strains’ genotypes were identified, all of which are within protein-coding genes of variable metabolic function. Single nucleotide polymorphisms (SNPs) provided the most reliable genetic markers, and amongst 55 strains of S. marinoi, three 500 bp loci contained, on average, 46 SNPs, 103 unique alleles, and displayed 100% heterozygosity. The prevalence of heterozygosity was identified as a novel opportunity to improve strain quantification and detect false positive artefacts during denoising of amplicon sequences. Finally, we illustrate how metabarcoding of a single genetic locus can be used to track strain abundances of 58 strains of S. marinoi in an artificial selection experiment. As future genomics datasets become available and DNA sequencing technologies develop, Bamboozle has flexible user settings enabling optimal barcodes to be designed for other species and applications.

Bamboozle: A bioinformatic tool for identification and quantification of intraspecific barcodes