Sequencing & Pangenomes

Amp-Seq One, a Single Reaction Targeted Genotyping Chemistry for High Throughput Applications

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Abstract

Targeted genotyping remains a central tool for breeders and researchers to inform selection efforts, allowing for rapid advancements in genetic gains. Multiplexed PCR-based genotyping, coupled with Next Generation Sequencing, has proven to be an effective tool that provides advantages in flexibility and scale over traditional technologies. However, multiplexed PCR reactions can suffer from elevated sample to sample variation, inconsistent locus recovery, and wide variability in sequencing reads per locus. Here, we present a chemistry that helps address the shortcomings of PCR-based genotyping assays. Amp-Seq One is a single reaction workflow where sample DNA is combined with targeting oligos and master-mix into a single use 384 well reaction plate containing dried down illumina indexing oligos. Following thermocycling, samples are pooled by volume, bead cleaned, and are ready for loading onto a sequencer. Here, we present performance data from panels in bovine and row-crops ranging in size from 50 to 5200 loci. These include 2 panels targeting the bovine standard ISAG (199 loci) and ICAR (554 loci) maker sets. Targeting oligos were designed optimizing for uniqueness within the genome, uniform TM & GC content, and reduced primer-primer interaction potential. Genotype call rates per sample for all panels were high, ranging from 90 - 99%, with >99% for the ISAG panel and >98% for the ICAR panel. Locus-to-locus variability, measured as uniformity, was well controlled with >80% of loci having depths greater than 0.2x of the mean depth, with the ISAG and ICAR panels showing >95%. This chemistry also efficiently utilizes each sample's sequencing data with most panels showing >90% of all sequencing reads generated mapping to their intended targets within the genome. While the libraries are generated with illumina based indexes and adaptors, similar performance was observed when libraries were sequenced on an Element Aviti and MGI platforms, following appropriate library conversion. The Amp-Seq One chemistry provides a flexible solution for customizable panels that provides a high throughput tool for breeders capable of returning consistent locus recovery, efficient use of sequencing data through high on-target rates, and favorable locus to locus variability within a sample.

Keywords: 2026

How to Cite:

Payton, A., Walker, J. & Jasinovica, S., (2026) “Amp-Seq One, a Single Reaction Targeted Genotyping Chemistry for High Throughput Applications”, World Congress on Genetics Applied to Livestock Production Digital Archive 2026(1): 2287227. doi: https://doi.org/10.31274/wcgalp.24240

Rights: 1