# A duplex sequencing approach for high-sensitivity detection of genome-edited plants

## TL;DR

This paper introduces a high-sensitivity sequencing method to detect small DNA changes in plants edited with new genomic techniques.

## Contribution

The study presents the first performance evaluation of a duplex sequencing approach for NGT-induced mutations without prior mutation knowledge.

## Key findings

- The method detected mutations as low as 0.1% in mixed samples.
- Duplex sequencing confirmed mutations on both DNA strands to avoid PCR and sequencing errors.
- The approach works without prior knowledge of mutation type or position.

## Abstract

In this paper, we have evaluated a targeted high-throughput massive parallel sequencing approach for detecting single nucleotide mutations or small genomic changes generated by new genomic techniques (NGT).

We used unique molecular identifiers (UMIs) for the quantification of the mutant alleles and duplex sequencing to confirm a mutation on both strands to avoid polymerase chain reaction (PCR) artefacts or sequencing miss-calls. We tested the approach in blinded analyses on a set of mixed NGT-modified tomato lines and identified each single nucleotide mutation or small insert/deletion (InDel) down to a 0.1 % level. To our knowledge, this is the first performance evaluation of a duplex sequencing approach for detecting and quantifying small NGT DNA changes without a priori knowledge of the mutation type and position in a target region.

Our study advances the scientific discussion on detecting NGT-induced DNA modifications in plants and food products, evaluating the potential and current limitations of a cutting-edge NGS-approach.

•Detection of mutations generated by new genomic techniques (NGT) is challenging.•We identified small NGT mutations using a targeted duplex-sequencing approach.•Each mutation was quantified down to a 0.1 % level also in mixed samples.•The approach does not require a priori knowledge of the exact mutation type.

Detection of mutations generated by new genomic techniques (NGT) is challenging.

We identified small NGT mutations using a targeted duplex-sequencing approach.

Each mutation was quantified down to a 0.1 % level also in mixed samples.

The approach does not require a priori knowledge of the exact mutation type.