Campbell, N. R.; Campbell, A. R.; Blair, S. K.; Finger, A. J.

GT-seq (Genotyping-in-Thousands by Sequencing) is widely used for high-throughput amplicon genotyping, but most analytical pipelines focus on single SNPs or rely on alignment-based variant calling. Here we present an alignment-free approach for microhaplotype genotyping that leverages the high read depth and low error rates typical of paired-end Illumina and Element sequencing. The pipeline first identifies primer-bounded reads and resolves paired-end sequences into complete amplicon sequences. Within each sample and locus, unique sequences are ranked by read abundance and the top one or two sequences are retained as candidate diploid alleles. These alleles are aggregated across samples to construct a catalog of unique haplotypes for each locus. In a second pass, reads are assigned to catalog haplotypes by exact sequence matching to produce diploid genotypes. Finally, catalog haplotype sequences are positionally compared to identify phased SNP and collapsed indel variation, generating compact microhaplotype representations suitable for population genetic analysis. This approach enables robust, alignment-free microhaplotype inference directly from high-depth amplicon sequencing data.