# A novel method for effectively selecting fragments not associated with restriction sites for whole-genome genotyping

## TL;DR

A new method called iRAD-seq simplifies and improves the efficiency of whole-genome genotyping by changing the order of library preparation and fragment selection.

## Contribution

The novel iRAD-seq method introduces a 'prepare library first, then select' workflow that streamlines RAD-seq library preparation.

## Key findings

- iRAD-seq provides consistent genome-wide SNP distributions in maize and rice.
- The method enables effective genetic diversity analysis in maize germplasm.
- iRAD-seq is useful for identifying quantitative trait loci in maize populations.

## Abstract

Reduced representation sequencing (RRS) using Restriction site-associated DNA sequencing (RAD-seq) has become a widely adopted method for whole-genome genotyping, owing to its cost-effectiveness and applicability across species. However, traditional RAD-seq approaches face challenges, including complex workflows and high labor demands. Existing RAD-seq methods rely primarily on the strategy of “selecting fragments first, then preparing a library,” which involves targeting fragments associated with restriction enzyme cut sites.

In contrast, we developed inverse RAD-seq (iRAD-seq), a novel method that employs a “prepare library first, then select” strategy to efficiently capture fragments not associated with restriction sites for whole-genome genotyping. This innovative approach utilizes Tn5 transposase to simultaneously fragment DNA and ligate adapters, followed by pooled processing of hundreds of libraries for batch restriction digestion, thereby significantly streamlining RAD-seq library preparation. This simplified workflow makes iRAD-seq highly compatible with liquid handling automation, enhancing its throughput. We validated iRAD-seq through both in silico analyses and wet-lab experiments in maize and rice. The results demonstrate that iRAD-seq provides consistent genome-wide Single-Nucleotide Polymorphism (SNP) distributions. In maize germplasm, iRAD-seq successfully enables effective genetic diversity analysis. Furthermore, genetic mapping of maize populations has confirmed its utility for identifying quantitative trait loci (QTL).

Our findings suggest that iRAD-seq offers a more streamlined, efficient, and flexible approach to genotyping and marker development than traditional RAD-seq methods. Owing to its potential for high-throughput applications and cost reductions, iRAD-seq is a valuable tool for genomic research and molecular breeding.

The online version contains supplementary material available at 10.1186/s12915-025-02330-8.