K-seq, an affordable, reliable, and open Klenow NGS-based genotyping technology

Peio Ziarsolo,Victor Garcia-Carpintero,Joaquin Cañizares,Jose Blanca,Aureliano Bombarely,Rebeca Hilario,Tomas Hasing

doi:10.1186/s13007-021-00733-6

Abstract

BackgroundK-seq, a new genotyping methodology based on the amplification of genomic regions using two steps of Klenow amplification with short oligonucleotides, followed by standard PCR and Illumina sequencing, is presented. The protocol was accompanied by software developed to aid with primer set design.ResultsAs the first examples, K-seq in species as diverse as tomato, dog and wheat was developed. K-seq provided genetic distances similar to those based on WGS in dogs. Experiments comparing K-seq and GBS in tomato showed similar genetic results, although K-seq had the advantage of finding more SNPs for the same number of Illumina reads. The technology reproducibility was tested with two independent runs of the tomato samples, and the correlation coefficient of the SNP coverages between samples was 0.8 and the genotype match was above 94%. K-seq also proved to be useful in polyploid species. The wheat samples generated specific markers for all subgenomes, and the SNPs generated from the diploid ancestors were located in the expected subgenome with accuracies greater than 80%.ConclusionK-seq is an open, patent-unencumbered, easy-to-set-up, cost-effective and reliable technology ready to be used by any molecular biology laboratory without special equipment in many genetic studies.

Highlights

K-seq, a new genotyping methodology based on the amplification of genomic regions using two steps of Klenow amplification with short oligonucleotides, followed by standard PCR and Illumina sequencing, is presented
Oligonucleotide selection and K‐seq library construction The short oligonucleotides used for the different species were selected by using a software application developed for this purpose, Primer Explorer
Those were combined to create potential oligonucleotide sets comprising 10 k-mers that were further analysed to check for compatibility in a PCR

Summary

Introduction

K-seq, a new genotyping methodology based on the amplification of genomic regions using two steps of Klenow amplification with short oligonucleotides, followed by standard PCR and Illumina sequencing, is presented. The NGS revolution has induced a very rapid improvement in genotyping technologies; it is standard to design experiments with hundreds of thousands of markers. This progress has accelerated gene and QTL analyses and allowed the popularisation of robust association studies as well as a marked increase in the scope of population diversity and evolution research in both model and non-model species. The genotyping cost per sample is usually low for these technologies, but they involve an initial costly SNP discovery and design step that must be paid upfront; so frequently, they are only available for model species.

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