Direct microhaplotype genotyping for GT-seq (Genotyping-in-Thousands by Sequencing) using a diploid abundance model
- 1GTseek LLC, Twin Falls, Idaho, United States of America
- 2University of California – Davis, Davis, California, United States of America
Abstract
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 a direct microhaplotype genotyping framework 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 quality-aware consensus amplicon sequences. Within each sample and locus, unique sequences are ranked by read abundance and the top one or two sequences are retained as directly observed haplotypes. These alleles are aggregated across samples to construct a catalog of observed 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 compared to identify phased SNP and collapsed indel variation. Optionally, catalog haplotypes may be aligned to a reference genome to project observed variants onto genomic coordinates and generate standards-compliant VCF output. This framework enables robust, microhaplotype genotyping directly from high-depth amplicon sequencing data. Comparison with an independent BWA/BCFtools alignment-based workflow demonstrated 99.67% genotype concordance across 102,520 genotype comparisons spanning 1,085 SNPs in 96 individuals. Genotype concordance remained above 99.4% even at the minimum supported sequencing depth of 10 reads per locus, demonstrating robust performance across a broad range of sequencing depths. Author summary: GT-seq has become a widely adopted method for genotyping hundreds of genetic markers across thousands of individuals because it combines low cost with high throughput. Existing analytical workflows commonly genotype predefined SNP or haplotype targets within each amplicon rather than reconstructing the complete amplicon sequence before identifying polymorphisms. We developed an open-source analysis framework that instead reconstructs complete amplicon haplotypes directly from paired-end sequencing data before identifying individual polymorphisms. This haplotype-first approach generates phased multi-allelic microhaplotypes without requiring conventional alignment-based variant calling while remaining compatible with standard VCF-based workflows. Using GT-seq data from a 410-locus panel, we demonstrate greater than 99.6% genotype concordance with an independent alignment-based pipeline and show that treating GT-seq amplicons as microhaplotypes increases mean marker information content by approximately 42% compared with single-SNP analysis. Because the method operates entirely in software, many existing GT-seq panels and datasets can be reanalyzed immediately as microhaplotypes without changes to laboratory protocols, providing a practical way to increase the value of previously generated sequencing data.
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BibTeX
@article{campbell2026direct,
title = {{Direct microhaplotype genotyping for GT-seq (Genotyping-in-Thousands by Sequencing) using a diploid abundance model}},
author = {Nathan R. Campbell and Amanda R. Campbell and Shannon K. Blair and Amanda J. Finger},
journal = {PLOS Computational Biology},
year = {2026},
month = sep,
doi = {10.1371/journal.pcbi.1014808},
url = {https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1014808},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Direct microhaplotype genotyping for GT-seq (Genotyping-in-Thousands by Sequencing) using a diploid abundance model
AU - Nathan R. Campbell
AU - Amanda R. Campbell
AU - Shannon K. Blair
AU - Amanda J. Finger
PY - 2026
DA - 2026/09/18
JO - PLOS Computational Biology
DO - 10.1371/journal.pcbi.1014808
UR - https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1014808
AB - 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 a direct microhaplotype genotyping framework 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 quality-aware consensus amplicon sequences. Within each sample and locus, unique sequences are ranked by read abundance and the top one or two sequences are retained as directly observed haplotypes. These alleles are aggregated across samples to construct a catalog of observed 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 compared to identify phased SNP and collapsed indel variation. Optionally, catalog haplotypes may be aligned to a reference genome to project observed variants onto genomic coordinates and generate standards-compliant VCF output. This framework enables robust, microhaplotype genotyping directly from high-depth amplicon sequencing data. Comparison with an independent BWA/BCFtools alignment-based workflow demonstrated 99.67% genotype concordance across 102,520 genotype comparisons spanning 1,085 SNPs in 96 individuals. Genotype concordance remained above 99.4% even at the minimum supported sequencing depth of 10 reads per locus, demonstrating robust performance across a broad range of sequencing depths. Author summary: GT-seq has become a widely adopted method for genotyping hundreds of genetic markers across thousands of individuals because it combines low cost with high throughput. Existing analytical workflows commonly genotype predefined SNP or haplotype targets within each amplicon rather than reconstructing the complete amplicon sequence before identifying polymorphisms. We developed an open-source analysis framework that instead reconstructs complete amplicon haplotypes directly from paired-end sequencing data before identifying individual polymorphisms. This haplotype-first approach generates phased multi-allelic microhaplotypes without requiring conventional alignment-based variant calling while remaining compatible with standard VCF-based workflows. Using GT-seq data from a 410-locus panel, we demonstrate greater than 99.6% genotype concordance with an independent alignment-based pipeline and show that treating GT-seq amplicons as microhaplotypes increases mean marker information content by approximately 42% compared with single-SNP analysis. Because the method operates entirely in software, many existing GT-seq panels and datasets can be reanalyzed immediately as microhaplotypes without changes to laboratory protocols, providing a practical way to increase the value of previously generated sequencing data.
SN - 1553-7358
ER -