Detection Of Rare Mutations Research Articles

EP.06F.05 External Quality Assessment Results of Third-party NGS Laboratories in China for the Detection of Rare Mutations in Lung Cancer

EP.06F.05 External Quality Assessment Results of Third-party NGS Laboratories in China for the Detection of Rare Mutations in Lung Cancer

Lab-In-Fiber Optofluidic Device for Droplet Digital Polymerase Chain Reaction (DdPCR) with Real-Time Monitoring.

Droplet microfluidic systems have emerged as indispensable and advanced tools in contemporary biological science. A prominent example is the droplet digital polymerase chain reaction (ddPCR), which plays a pivotal role in next-generation sequencing and the detection of rare nucleic acids or mutations. However, existing optical detection configurations are bulky, intricate, and costly, and require meticulous optical alignment to optimize fluorescence sensing. Herein, we propose a lab-in-fiber optofluidic system (LiFO), which provides a stable and compact footprint, self-alignment, and enhanced optical coupling for high-accuracy ddPCR. Moreover, LiFO could expand its capabilities for multiangle-scattering light collection in which we collect focused forward-scattering light (fFSL) to enable real-time droplet counting and size monitoring. To accomplish these attributes, LiFO incorporates optical fibers, along with fabricated PDMS grooves, for a self-aligned optical setup to implement simultaneous fluorescence and scattering detection. Furthermore, LiFO harnesses the concept of flowing droplets functioning as microlenses, which allows us to collect and translate fFSL signals into droplet size information. We have demonstrated the effectiveness of LiFO in ddPCR applications, illustrating its capacity to enhance the accuracy and precision of DNA quantification. Notably, LiFO exhibits improved linearity in the measurement of serial DNA dilutions, reflected by an increase in R2 from 0.956 to 0.997. These results demonstrate the potential of LiFO to serve as a valuable tool across a wide spectrum of droplet microfluidic platforms, offering opportunities for advancement in practical applications.

Loss of Heterozygosity and Mutations in the RAS-ERK Pathway Genes in Tumor Cells of Various Loci in Multiple Myeloma.

Multiple myeloma (MM) is a disease characterized by spatiotemporal heterogeneity of tumor clones. Different genetic aberrations can be observed simultaneously in tumor cells from different loci, and as the disease progresses, new subclones may appear. The role of liquid biopsy, which is based on the analysis of tumor DNA circulating in the blood plasma, continues to be explored in MM. Here, we present an analysis of the STR profiles and mutation status of the KRAS, NRAS, and BRAF genes, evaluated in plasma free circulating tumor DNA (ctDNA), CD138+ bone marrow cells, and plasmacytomas. The prospective single-center study included 97 patients, with a median age of 55 years. Of these, 94 had newly diagnosed symptomatic MM, and three had primary plasma cell leukemia. It should be noted that if mutations were detected only in ctDNA, "non-classical" codons were more often affected. A variety of adverse laboratory and clinical factors have been associated with the detection of rare KRAS or NRAS gene mutations in bone marrow or ctDNA, suggesting that these mutations may be factors of an unfavorable prognosis for MM. Liquid biopsy studies provide undeniable fundamental information about tumor heterogeneity and clonal evolution in MM. Moreover, we focus on using liquid biopsy to identify new high-risk factors for MM.

Methods and applications of genome-wide profiling of DNA damage and rare mutations.

DNA damage is a threat to genome integrity and can be a cause of many human diseases, owing to either changes in the chemical structure of DNA or conversion of the damage into a mutation, that is, a permanent change in DNA sequence. Determining the exact positions of DNA damage and ensuing mutations in the genome are important for identifying mechanisms of disease aetiology when characteristic mutations are prevalent and probably causative in a particular disease. However, this approach is challenging particularly when levels of DNA damage are low, for example, as a result of chronic exposure to environmental agents or certain endogenous processes, such as the generation of reactive oxygen species. Over the past few years, a comprehensive toolbox of genome-wide methods has been developed for the detection of DNA damage and rare mutations at single-nucleotide resolution in mammalian cells. Here, we review and compare these methods, describe their current applications and discuss future research questions that can now be addressed.

Advancements in the Detection and Treatment of Rare ALK Fusion Mutations in Hepatocellular Carcinoma: A Case Report and Literature Review

This case report and comprehensive literature review highlights the significance of advanced molecular diagnostic techniques, particularly next-generation sequencing (NGS), in the identification and treatment of rare ALK fusion mutations in hepatocellular carcinoma (HCC). Through a detailed analysis of a single patient case, accompanied by a review of existing literature, we underscore the diagnostic challenges and therapeutic potential associated with rare ALK fusions in HCC. Our findings demonstrate the superior capability of NGS, both DNA-based and RNA-based, in detecting these mutations, leading to targeted treatment approaches with ALK inhibitors. The case report illustrates the practical application of precision oncology in HCC, showcasing significant clinical benefit and improved treatment outcomes with the use of ALK inhibitors for rare fusion types. This study not only contributes to the existing body of knowledge by documenting a rare instance of ALK fusion in HCC but also advocates for the integration of comprehensive molecular profiling into standard clinical practice to enhance personalized treatment strategies and patient care.

Mismatch-Guided Deoxyribonucleic Acid Assembly Enables Ultrasensitive and Multiplex Detection of Low-Allele-Fraction Variants in Clinical Samples.

Somatic mutations are important signatures in clinical cancer treatment. However, accurate detection of rare somatic mutations with low variant-allele frequencies (VAFs) in clinical samples is challenging because of the interference caused by high concentrations of wild-type (WT) sequences. Here, we report a post amplification SNV-specific DNA assembly (PANDA) technology that eliminates the high concentration pressure caused by WT through a mismatch-guided DNA assembly and enables the ultrasensitive detection of cancer mutations with VAFs as low as 0.1%. Because it generates an assembly product that only exposes a single-stranded domain with the minimal length for signal readout and thus eliminates possible interferences from secondary structures and cross-interactions among sequences, PANDA is highly versatile and expandable for multiplex testing. With ultrahigh sensitivity, PANDA enabled the quantitative analysis of EGFR mutations in cell-free DNA of 68 clinical plasma samples and four pleuroperitoneal fluid samples, with test results highly consistent with NGS deep sequencing. Compared to digital PCR, PANDA returned fewer false negatives and ambiguous cases of clinical tests. Meanwhile, it also offers much lower upfront instrumental and operational costs. The multiplexity was demonstrated by developing a 3-plex PANDA for the simultaneous analysis of three EGFR mutations in 54 pairs of tumor and the adjacent noncancerous tissue samples collected from lung cancer patients. Because of the ultrahigh sensitivity, multiplexity, and simplicity, we anticipate that PANDA will find wide applications for analyzing clinically important rare mutations in diverse devastating diseases.

A Digital PCR Method Based on Highly Specific Taq for Detecting Gene Editing and Mutations.

Digital PCR (dPCR) has great potential for assessing gene editing or gene mutation due to its ability to independently inspect each DNA template in parallel. However, current dPCR methods use a fluorescence-labeled probe to detect gene variation events, and their ability to distinguish variated sequences from the wild-type sequence is limited by the probe's tolerance to mismatch. To address this, we have developed a novel dPCR method that uses a primer instead of a probe to sense gene variation. The enhanced Taq DNA polymerase in the PCR system has a high mismatch sensitivity, which enables our dPCR method to distinguish gene mutations from wild-type sequences. Compared to current dPCR methods, our method shows superior precision in assessing gene editing efficiency and single-base DNA mutation. This presents a promising opportunity to advance gene editing research and rare gene mutation detection.

Dominant Optic Atrophy With Varied Phenotype in a Family of Eastern India: A Case Report of a Rare Mutation

Background: Autosomal dominant optic atrophy (ADOA) can present with a varied phenotype in the form of pure ADOA, ADOA with sensorineural hearing loss, or the extended spectrum of DOA plus syndrome. Multiple genes have been identified over the years and majority of the cases are attributed to OPA1 gene mutation. Here we present an index case of DOA plus syndrome with affection of multiple family members across 2 generations with a rare mutation detection. Case Presentation: A middle-aged female presented with onset of bilateral progressive visual blurring in the first decade followed by progressive sensorineural hearing loss since the second decade and onset of lower limb predominant cerebellar ataxia since the third decade. Routine parameters, imaging and electrophysiological studies were negative. Next generation clinical exome sequencing (NGCES) revealed an unique OPA1 gene mutation with worldwide evidence of only 3 such cases in database. Conclusion: DOA suspicion should be made in early onset visual defects with multiaxial involvement with a significant family history.

Detection of rare mutations, copy number alterations, and methylation in the same template DNA molecules

The analysis of cell-free DNA (cfDNA) from plasma offers great promise for the earlier detection of cancer. At present, changes in DNA sequence, methylation, or copy number are the most sensitive ways to detect the presence of cancer. To further increase the sensitivity of such assays with limited amounts of sample, it would be useful to be able to evaluate the same template molecules for all these changes. Here, we report an approach, called MethylSaferSeqS, that achieves this goal, and can be applied to any standard library preparation method suitable for massively parallel sequencing. The innovative step was to copy both strands of each DNA-barcoded molecule with a primer that allows the subsequent separation of the original strands (retaining their 5-methylcytosine residues) from the copied strands (in which the 5-methylcytosine residues are replaced with unmodified cytosine residues). The epigenetic and genetic alterations present in the DNA molecules can then be obtained from the original and copied strands, respectively. We applied this approach to plasma from 265 individuals, including 198 with cancers of the pancreas, ovary, lung, and colon, and found the expected patterns of mutations, copy number alterations, and methylation. Furthermore, we could determine which original template DNA molecules were methylated and/or mutated. MethylSaferSeqS should be useful for addressing a variety of questions relating genetics and epigenetics.

Abstract 6609: Detection of rare mutations, copy number variation, and DNA methylation in the same template DNA molecules

Abstract The analysis of cell-free DNA (cfDNA) from plasma offers great promise for the earlier detection of cancer. At present, changes in DNA sequence, methylation, or copy number are the most sensitive ways to detect the presence of cancer. To increase the sensitivity of such assays, it would be useful to be able evaluate the same template molecules for all these changes. Here we report an approach, called MethylSaferSeqS, that achieves this goal, and can be applied to any standard library preparation method suitable for massively parallel sequencing. The innovative step was to copy both strands of each DNA-barcoded molecule with a primer that allows the subsequent separation of the original strands (retaining their 5-methylcytosine residues) from the copied strands (in which the 5-methylcytosine residues are replaced with unmodified cytosine residues). The original and copied strands are queried for epigenetic and genetic alterations, respectively. We applied this approach to plasma from 265 individuals, including 198 with cancers of the pancreas, ovary, lung or colon, and found the expected patterns of mutations, copy number alterations, and methylation. Furthermore, we could determine which original template DNA molecules were methylated and/or mutated. MethylSaferSeqS should be useful for addressing a variety of questions relating genetics and epigenetics in the future. Citation Format: Yuxuan Wang, Christopher Douville, Joshua Cohen, Chetan Bettegowda, Nick Papadopoulos, Ken Kinzler, Bert Vogelstein. Detection of rare mutations, copy number variation, and DNA methylation in the same template DNA molecules. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6609.

Abstract 6681: XNA increases assay sensitivity in sanger sequencing, qPCR, NGS and CRISPR mutant screening

Abstract The proprietary XNA (xeno nucleic acid) technology has wide applications in sensitive detection of rare mutations in different genomic platforms. These target-specific DNA oligo analogs increase assay sensitivity by blocking the polymerase-dependent amplification of wildtype sequence so only amplification of the mutant sequence occurs. Here we show that the XNA technology helps enrich rare somatic mutations in different human specimen types such as FFPE and plasma samples, using technology platforms such as Sanger sequencing, TaqMan qPCR, and NGS. XNA increases assay sensitivity to 0.1% for Sanger sequencing and qPCR. For NGS, XNA allows detection of the mutant sequence at a lower sequencing depth, thus combining XNA technology with NGS for MRD (molecular residual disease) detection could improve cancer management. In addition, We also show CRISPR mutant screening using a target-specific XNA in a SYBR Green qPCR assay. XNA can be used for rapid CRISPR mutant screening for DNA from pool or individual clones generated from gene editing experiments. In summary, XNA technology is a powerful tool when combined with existing genomic mutation detection platforms to screen for rare somatic mutations and for rapid CRISPR mutant screening. The technology is customizable as a service for easy integration with current technology platforms and applications. Citation Format: Wei Liu, Robert Brown, Andrew Fu, Shuo Shen, Michael Sha, Aiguo Zhang. XNA increases assay sensitivity in sanger sequencing, qPCR, NGS and CRISPR mutant screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6681.

Improving the Accuracy of Single-Nucleotide Variant Diagnosis Using On–Off Discriminating Primers

Early detection of rare mutations through liquid biopsy can provide real-time information related to cancer diagnosis, prognosis, and treatment outcomes. Cell-free DNA samples used in liquid biopsies contain single-nucleotide variants (SNVs) with a variant allele frequency (VAF) of approximately ≤1%. Droplet digital polymerase chain reaction (ddPCR) is considered the gold standard of sequencing using liquid samples, generating amplicons from samples containing mutations with 0.001-0.005% VAF; however, it requires expensive equipment and time-consuming protocols. Therefore, various PCR methods for discriminating SNVs have been developed; nonetheless, non-specific amplification cannot be avoided even in the absence of mutations, which hampers the accurate diagnosis of SNVs. In this study, we introduce single-nucleotide variant on-off discrimination-PCR (Soo-PCR), a highly accurate and practical method that uses a 3'-end tailing primer for the on-off discrimination of low-abundance mutant-type targets, including SNVs. Soo-PCR minimizes the chance of incorrect judgments owing to its high discriminating power. Cancer markers, such as KRAS G12D, EGFR L858R, and EGFR T790M mutations, containing 0.1% VAF, were clearly detected in under 2 h with a high reliability comparable with that of ddPCR. This new method serves as a practical approach to accurately detect and evaluate low-abundance mutations in a user-friendly manner.

Identification of a Novel 7-bp Deletion in the α-Globin Gene Cluster in One Chinese Family

Deletional α-thalassemia is characterized by reduced hemoglobin A2 and involves the deletion of a few nucleotides, which is a rare hereditary disease. However, the detection of rare mutations using commonly used genetic tests is highly challenging. In the present study, next-generation sequencing (NGS) was used to identify a novel 7-bp deletion α-thalassemia in one individual from a Chinese family. Hematological parameters of the family members were determined using an automated cell counter, and hemoglobin electrophoresis was performed using a capillary electrophoresis system. Subsequently, NGS was performed on the genomic DNA of the patient and her family members. The 7-bp deletion (named Hb Honghe [HBA1: c.401_407delGCACCGT]) of α-thalassemia in the α-globin gene was confirmed using Sanger sequencing. The patient’s father was also a heterozygous carrier of HBA1: c.401_407delGCACCGT deletion, but not her mother or sister. The application of the combined molecular approach is essential for the accurate diagnosis of rare thalassemia. This study reports a novel case of α- thalassemia. The characterization of the mutation might provide new insights into genetic counseling and accurate diagnosis of thalassemia.

In vitro Argonaute cleavage-mediated quantitative PCR facilitates versatile CRISPR/Cas-induced mutant analysis

CRISPR/Cas9 and associated base editing systems are used to precisely modify target genes in diverse cell types and organisms. However, CRISPR/Cas-induced mutants often contain small insertions and deletions (indels) or single nucleotide variations (SNVs) at edited sites with different genotypes, and gene editing frequency (GEF) typically varies between editing sites and editing systems. Therefore, methods are needed to identify mutants and their genotypes, and evaluate GEF. Herein, we describe a novel SMART approach for simultaneous analysis of CRISPR/Cas-induced mutants, genotypes and GEF, using DNA-guided Thermus thermophilus Argonaute (TtAgo) in vitro cleavage-mediated quantitative real-time PCR (qPCR) or digital PCR. SMART proved versatile for induced mutant identification, genotyping, and GEF evaluation using CRISPR/Cas9 gene-editing offspring lines harbouring the rice RNA-directed DNA methylation 3-like (RdDM3l) gene. SMART is higher specific, sensitive, and accurate than previously methods. SMART detected all types of mutations, including small indels, large indels and nucleotide substitutions, with good sensitivity regardless of mutation type. TtAgo has few strict requirements or restrictions for target sequences, such as restriction sites, GC content or protospacer-adjacent motifs (PAMs). SMART facilitates versatile CRISPR/Cas-induced mutant screening, genotyping, and GEF quantification, and it could be developed for clinical detection of rare mutations involving SNVs and indels.

Validation of a highly sensitive Sanger sequencing in detecting EGFR mutations from circulating tumor DNA in patients with lung cancers

BackgroundThe novel method, named blocker displacement amplification (BDA) Sanger, was applied to detect low variant allele frequency mutations in the circulating tumor DNA (ctDNA). This study aimed to evaluate the performance of the BDA Sanger method for the EGFR mutation detection in the ctDNA from lung cancer patients. MethodsA total of 195 plasma samples of lung cancer patients were included. The EGFR mutation status in the ctDNA was detected by the BDA Sanger and Super-ARMS assays. Next-generation sequencing (NGS) was further used to verify the mutant of EGFR with inconsistencies. ResultsBDA Sanger assay was capable of detecting EGFR mutations with a 0.20% VAF from plasma samples. Among treatment-naive patients with paired tissue and plasma samples, the EGFR positive percent agreement (PPA) was 79% by BDA sanger. EGFR mutation was detected in 34.4% (67/195) ctDNA samples by the Super-ARMS and in 41.0% (80/195) ctDNA samples by the BDA Sanger assay. The overall concordance rate between the BDA Sanger and Super-ARMS assays was 82% (160/195). The BDA Sanger also enabled the detection of rare EGFR mutations, which were not discovered by the Super-ARMS. ConclusionThe results supported the validity and efficiency of the BDA Sanger method for EGFR detection in patients with lung cancer, indicating that BDA Sanger has a great potential for application in detecting mutations in the ctDNA.

Abstract 763: Linking Down syndrome, clonal hematopoiesis, and autophagy

Abstract Down syndrome (DS) is the result of trisomy in chromosome 21 (T21) and is associated with an increased risk of diseases, particularly acute myeloid leukemia (AML). However, people with DS have variable disease risks and severities that have yet to be explained. We hypothesize that differing characteristics of clonal hematopoiesis (CH) may coincide with and/or contribute to poor health outcomes seen in people with DS and can help explain differences in disease outcome. To characterize CH, we use a rare-mutation detection technique called Duplex Seq, only requiring a blood draw to obtain the DNA from leukocytes. This targeted-sequencing tool uses a double-stranded tag to incorporate mutational data from both strands of DNA, enabling high sensitivity through the elimination of DNA processing errors. Using bioinformatics and COSMIC’s Cancer Gene Census, we have established differences in mutational patterns that enable us to distinguish people with DS from people without DS and characterize the variability among people with DS. Notably, protein-altering mutations in genes responsible for the differentiation of hematopoietic stem and progenitor cells are more prevalent among people with T21 than their counterparts who are disomic in chromosome 21 (D21). Coupling our genomic findings with other -omics approaches, we have drawn associations between the observed mutations and phenotypes of these individuals. Using mouse models of DS (Dp16, Dp17, and Dp10), we plan to investigate the susceptibility of CH and leukemogenesis with the use of a Moloney Murine Leukemia virus, expecting the mice modeling DS to have more prevalent CH and more leukemias. Furthermore, based on our analyses of leukocytic gene expression differences between people with DS with or without CH, we believe that people with DS have a varying reduction in autophagy that enables the presence of harmful CH and increased disease risk in some individuals. Using a GFP-LC3-RFP construct, we plan to quantify and compare basal autophagic flux between the mouse models of DS and their control littermates. This will enable us to draw the links between DS, CH, and autophagy. Future work will be centered around manipulating autophagy using transgenic mice and non-invasive interventions to see if deleterious phenotypes and risks to AML can be reversed. Overall, these studies offer a means by which disease risk in people with DS can be monitored through CH and a potential mechanism that can be altered by non-intrusive means to improve longevity and quality of life. Citation Format: Edward James Evans, Ross Granrath, Keith Smith, Joaquin Espinosa, Andrew Thorburn, James DeGregori. Linking Down syndrome, clonal hematopoiesis, and autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 763.

Detection of rare thalassemia mutations using long-read single-molecule real-time sequencing.

Gap- polymerase chain reaction (PCR), reverse dot-blot assay (RDB), real-time PCR based multicolor melting curve analysis (MMCA assay), multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing are conventional methods to diagnose thalassemia but all of them have limitations. In this study, we applied single-molecule real-time (SMRT) sequencing following multiplex long-range PCR to uncover rare mutations in nine patients and their family members. The patients with different results between Gap-PCR and MMCA assay or with phenotype not matching genotype were included. Using SMRT sequencing, we first identified the carriers with αααanti3.7/HKαα, -α762bpα/αα (chr16:172,648-173,409), ααfusion/αQSα (in a trans configuration), two cases with novel gene rearrangements and another case with a novel 341bp insertion in α-globin gene cluster, respectively. One carrier with --SEA/αααanti4.2, and two carriers with the coexistence of globin variant and an α-globin gene duplication were also found. Most importantly, we could determine two defects in α-globin gene cluster being a cis or trans configuration in a single test. Our results showed that SMRT has great advantages in detection of α-globin gene triplications, rare deletions and determination of a cis or trans configuration. SMRT is a comprehensive and one-step method for thalassemia screening and diagnosis, especially for detection of rare thalassemia mutations.

Microfluidic cell sorter sample preparation for genomic assays.

Single-cell RNA-Sequencing has led to many novel discoveries such as the detection of rare cell populations, microbial populations, and cancer mutations. The quality of single-cell transcriptomics relies heavily on sample preparation and cell sorting techniques that best preserve RNA quality while removing dead cells or debris prior to cDNA generation and library preparation. Magnetic bead cell enrichment is a simple process of cleaning up a sample but can only separate on a single-criterion. Droplet-based cell sorters, on the other hand, allows for higher purity of sorted cells gated on several fluorescent and scatter properties. The downside of traditional droplet-based sorters is their operational complexity, accessibility, and potential stress on cells due to their high-pressure pumps. The WOLF® Cell Sorter, and WOLF G2®, developed by NanoCellect Biomedical, are novel microfluidic-based cell sorters that use gentle sorting technology compatible with several RNA-sequencing platforms. The experiments highlighted here demonstrate how microfluidic sorting can be successfully used to remove debris and unwanted cells prior to genomic sample preparation resulting in more data per cell and improved library complexity.

Multiplex SuperSelective PCR Assays for the Detection and Quantitation of Rare Somatic Mutations in Liquid Biopsies

SuperSelective primers, by virtue of their unique design, enable the simultaneous identification and quantitation of inherited reference genes and rare somatic mutations in routine multiplex PCR assays, while virtually eliminating signals from abundant wild-type sequences closely related to the target mutations. These assays are sensitive, specific, rapid, and low cost, and can be performed in widely available spectrofluorometric thermal cyclers. Herein, we provide examples of SuperSelective PCR assays that target eight different somatic EGFR mutations, irrespective of whether they occur in the same codon, occur at separate sites within the same exon, or involve deletions. In addition, we provide examples of SuperSelective PCR assays that detect specific EGFR mutations in circulating tumor DNA present in the plasma of liquid biopsies obtained from patients with non-small-cell lung cancer. The results suggest that multiplex SuperSelective PCR assays may enable the choice, and subsequent modification, of effective targeted therapies for the treatment of an individual's cancer, utilizing frequent noninvasive liquid biopsies.

Single-strand specific nuclease enhances accuracy of error-corrected sequencing and improves rare mutation-detection sensitivity

Error-corrected sequences (ECSs) that utilize double-stranded DNA sequences are useful in detecting mutagen-induced mutations. However, relatively higher frequencies of G:C > T:A (1 × 10−7 bp) and G:C > C:G (2 × 10−7 bp) errors decrease the accuracy of detection of rare G:C mutations (approximately 10−7 bp). Oxidized guanines in single-strand (SS) overhangs generated after shearing could serve as the source of these errors. To remove these errors, we first computationally discarded up to 20 read bases corresponding to the ends of the DNA fragments. Error frequencies decreased proportionately with trimming length; however, the results indicated that they were not sufficiently removed. To efficiently remove SS overhangs, we evaluated three mechanistically distinct SS-specific nucleases (S1 Nuclease, mung bean nuclease, and RecJf exonuclease) and found that they were more efficient than computational trimming. Consequently, we established Jade-Seq™, an ECS protocol with S1 Nuclease treatment, which reduced G:C > T:A and G:C > C:G errors to 0.50 × 10−7 bp and 0.12 × 10−7 bp, respectively. This was probably because S1 Nuclease removed SS regions, such as gaps and nicks, depending on its wide substrate specificity. Subsequently, we evaluated the mutation-detection sensitivity of Jade-Seq™ using DNA samples from TA100 cells exposed to 3-methylcholanthrene and 7,12-dimethylbenz[a]anthracene, which contained the rare G:C > T:A mutation (i.e., 2 × 10−7 bp). Fold changes of G:C > T:A compared to the vehicle control were 1.2- and 1.3-times higher than those of samples without S1 Nuclease treatment, respectively. These findings indicate the potential of Jade-Seq™ for detecting rare mutations and determining the mutagenicity of environmental mutagens.