Next-generation sequencing (NGS) has the potential to revolutionize clinical medicine and basic research, but its accuracy is limited by a 1% error rate, which can lead to false positives in detecting rare mutations. To address this, researchers developed Duplex Sequencing, a method that reduces errors by independently sequencing both strands of a DNA duplex. True mutations appear in both strands, while errors occur in only one. This approach reduces the background error rate to less than one artificial mutation per billion nucleotides sequenced. The study applied Duplex Sequencing to detect mutations in mitochondrial DNA from human cells, revealing a mutation frequency of 3.0 × 10⁻⁶, consistent with established genetic methods. The method was tested on M13mp2 DNA, a substrate with a known mutation frequency. Standard sequencing resulted in a high error rate, but Duplex Sequencing significantly reduced it. Analysis of the data showed that mutations detected by Duplex Sequencing were more accurate, with a mutation frequency of 2.5 × 10⁻⁶, nearly matching the reference value. The study also found that DNA damage, such as oxidative damage, can cause mutations that are difficult to distinguish from true mutations. However, Duplex Sequencing effectively identified and corrected these errors. The method was further validated by detecting rare mutations in M13mp2 variants, demonstrating its ability to identify mutations present at very low frequencies. When applied to human mitochondrial DNA, Duplex Sequencing revealed a much lower mutation frequency than standard sequencing, indicating its effectiveness in reducing technical errors. The method also identified mutational hotspots in the mitochondrial genome, particularly near the replication initiation region, which is consistent with prior findings. Duplex Sequencing offers a powerful tool for detecting rare mutations with high accuracy, enabling the identification of true mutations in complex genetic mixtures. It also allows for the detection of DNA damage sites, which is crucial for understanding the mutagenic effects of various factors. The method is applicable to a wide range of sequencing platforms and can be used for both error correction and precise molecular counting. Overall, Duplex Sequencing significantly improves the accuracy of next-generation sequencing, making it a valuable tool in clinical and research settings.Next-generation sequencing (NGS) has the potential to revolutionize clinical medicine and basic research, but its accuracy is limited by a 1% error rate, which can lead to false positives in detecting rare mutations. To address this, researchers developed Duplex Sequencing, a method that reduces errors by independently sequencing both strands of a DNA duplex. True mutations appear in both strands, while errors occur in only one. This approach reduces the background error rate to less than one artificial mutation per billion nucleotides sequenced. The study applied Duplex Sequencing to detect mutations in mitochondrial DNA from human cells, revealing a mutation frequency of 3.0 × 10⁻⁶, consistent with established genetic methods. The method was tested on M13mp2 DNA, a substrate with a known mutation frequency. Standard sequencing resulted in a high error rate, but Duplex Sequencing significantly reduced it. Analysis of the data showed that mutations detected by Duplex Sequencing were more accurate, with a mutation frequency of 2.5 × 10⁻⁶, nearly matching the reference value. The study also found that DNA damage, such as oxidative damage, can cause mutations that are difficult to distinguish from true mutations. However, Duplex Sequencing effectively identified and corrected these errors. The method was further validated by detecting rare mutations in M13mp2 variants, demonstrating its ability to identify mutations present at very low frequencies. When applied to human mitochondrial DNA, Duplex Sequencing revealed a much lower mutation frequency than standard sequencing, indicating its effectiveness in reducing technical errors. The method also identified mutational hotspots in the mitochondrial genome, particularly near the replication initiation region, which is consistent with prior findings. Duplex Sequencing offers a powerful tool for detecting rare mutations with high accuracy, enabling the identification of true mutations in complex genetic mixtures. It also allows for the detection of DNA damage sites, which is crucial for understanding the mutagenic effects of various factors. The method is applicable to a wide range of sequencing platforms and can be used for both error correction and precise molecular counting. Overall, Duplex Sequencing significantly improves the accuracy of next-generation sequencing, making it a valuable tool in clinical and research settings.