Sequencing small genomic targets with high efficiency and extreme accuracy.
paper
Cited
Public
- Authors
- Schmitt, Michael W; Fox, Edward J; Prindle, Marc J; Reid-Bayliss, Kate S; True, Lawrence D; Radich, Jerald P; Loeb, Lawrence A
- Year
- 2015
- Journal
- Nature methods
- PMID
- 25849638
- DOI
- 10.1038/nmeth.3351
- PMCID
- PMC4414912
The detection of minority variants in mixed samples requires methods for enrichment and accurate sequencing of small genomic intervals. We describe an efficient approach based on sequential rounds of hybridization with biotinylated oligonucleotides that enables more than 1-million-fold enrichment of genomic regions of interest. In conjunction with error-correcting double-stranded molecular tags, our approach enables the quantification of mutations in individual DNA molecules.
High on-target recovery with sequential rounds of capture. (a) Human genomic DNA captured with biotinylated probes targeting Abl exons 4β7 results in low on-target recovery following one round of capture, while two rounds result in >97% of reads mapping to the targeted gene. Experiment 1 was carried out with conventional blocking oligonucleotides mws60 and mws61; experiment 2 used chemically modified high-affinity blocking oligonucleotides mws58 and mws59. (b) Percent of targeted nucleotides covered at a given sequencing depth following single and double capture. Both samples were sequenced on an equivalent fraction of a HiSeq 2500 lane (5 Γ 106 paired-end reads, corresponding to 3% of a single lane).
LLM interpretation
Figure (a) is a stacked bar chart showing the percentage of on-target reads (targeted exons in light gray and flanking introns in dark gray) across one, two, and three rounds of capture for two different experiments. In both experiments, on-target recovery increases from <10% after one round to nearly 100% after two or three rounds. Figure (b) is a line graph plotting the percent of targeted nucleotides covered against sequencing depth, showing that double capture (red line) maintains significantly higher coverage at greater sequencing depths compared to single capture (blue line).
Removal of sequencing artifacts by Duplex Sequencing. (a) Exons in Abl spanning the active site of the enzyme were enriched by the double-capture protocol and sequenced conventionally on an Illumina HiSeq 2500. Despite extremely stringent quality filtering (minimum Phred score 50), and removal of end-repair artifacts by 5-nucleotide trimming from read ends, true mutations cannot be discerned among the thousands of sequencing errors that persist. (b) Duplex Sequencing of the same sample reveals a single point mutation in Abl which confers imatinib resistance. The mutation was verified by RT-PCR and Sanger sequencing.
LLM interpretation
This figure consists of two line plots (a and b) showing the mutant fraction across genome positions for the *Abl* gene. Panel (a), representing conventional sequencing, shows a high level of background noise with numerous peaks of mutant fractions reaching up to 2.5%. In contrast, panel (b), representing Duplex Sequencing, shows a flat baseline with a single distinct peak at approximately genome position 1000, indicating a single point mutation.
1β7 of 7
No entities extracted from this document yet.
No uploaded files.
Not in any collection.
In this knowledge base
| Title | Year | PMID |
|---|---|---|
| Statistical modeling for sensitive detection of low-frequency single nucleotide variants. | 2016 | 27556804 |
External
| Title | Authors | Journal | Year | Link |
|---|---|---|---|---|
| A targeted DNA methylation method for detecting gastrointestinal cancer in circulating cell-free DNA. | Jiang Z et al. | β | 2026 | β |
| Chemical tools for discriminating single nucleotide variants: from design principles to clinical applications. | Huang D et al. | β | 2026 | β |
| Supersensitive and robust disease monitoring in oropharyngeal cancer patients by circulating tumor HPV-DNA sequencing (ctHPV-DNAseq). | Pierik AS et al. | β | 2026 | β |
| Allele frequency selection and no age-related increase in human oocyte mitochondrial mutations. | Arbeithuber B et al. | β | 2025 | β |
| Correcting errors in PCR-derived libraries for rare allele detection by reconstructing parental and daughter strand information. | Lim H et al. | β | 2025 | β |
| De novo rates of a <i>Trypanosoma</i>-resistant mutation in two human populations. | Melamed D et al. | β | 2025 | β |
| Digital sequencing is improved by using structured unique molecular identifiers. | Micallef P et al. | β | 2025 | β |
| Enhanced Error Suppression for Accurate Detection of Low-Frequency Variants. | Chen H et al. | β | 2025 | β |
| Improved Quantification of Circulating Tumor DNA in Translocation-Associated Myxoid Liposarcoma by Simultaneous Detection of Breakpoints and Single Nucleotide Variants. | Schmid A et al. | β | 2025 | β |
| MAGNET-seq: A tandem PCR and hybrid capture method for enhanced target enrichment. | Lee D et al. | β | 2025 | β |
| Quantifying the mutational landscape of retroviral and lentiviral vectors in gene therapy patients. | Hart KL et al. | β | 2025 | β |
| Detection methods and prognosis implications of measurable residual disease in acute myeloid leukemia. | Zhao Z et al. | β | 2024 | β |
| High-Grade Serous Ovarian Cancer-A Risk Factor Puzzle and Screening Fugitive. | WilczyΕski J et al. | β | 2024 | β |
| Intratumor genetic heterogeneity and head and neck cancer relapse. | Pierik AS et al. | β | 2024 | β |
| Neutral evolution of rare cancer mutations in the computer and the clinic. | Beckman RA | β | 2024 | β |
| Somatic mutations in aging and disease. | Ren P et al. | β | 2024 | β |
| Subclonal Cancer Driver Mutations Are Prevalent in the Unresected Peritumoral Edema of Adult Diffuse Gliomas. | Underhill HR et al. | β | 2024 | β |
| DELFMUT: duplex sequencing-oriented depth estimation model for stable detection of low-frequency mutations. | Wu G et al. | β | 2023 | β |
| Next-generation sequencing methodologies to detect low-frequency mutations: "Catch me if you can". | Menon V et al. | β | 2023 | β |
| Non-invasive monitoring of neoadjuvant radiation therapy response in soft tissue sarcomas by multiparametric MRI and quantification of circulating tumor DNA-A study protocol. | Runkel A et al. | β | 2023 | β |
| Single duplex DNA sequencing with CODEC detects mutations with high sensitivity. | Bae JH et al. | β | 2023 | β |
| The changing face of circulating tumor DNA (ctDNA) profiling: Factors that shape the landscape of methodologies, technologies, and commercialization. | Bronkhorst AJ et al. | β | 2023 | β |
| Tracing Founder Mutations in Circulating and Tissue-Resident Follicular Lymphoma Precursors. | Schroers-Martin JG et al. | β | 2023 | β |
| Accurate detection of subclonal variants in paired diagnosis-relapse acute myeloid leukemia samples by next generation Duplex Sequencing. | Kamath-Loeb AS et al. | β | 2022 | β |
| Antiprogestins reduce epigenetic field cancerization in breast tissue of young healthy women. | Bartlett TE et al. | β | 2022 | β |
| Discovery of an unusually high number of de novo mutations in sperm of older men using duplex sequencing. | Salazar R et al. | β | 2022 | β |
| Genotyping of Circulating Free DNA Enables Monitoring of Tumor Dynamics in Synovial Sarcomas. | Eisenhardt AE et al. | β | 2022 | β |
| Individualized Mini-Panel Sequencing of ctDNA Allows Tumor Monitoring in Complex Karyotype Sarcomas. | Braig D et al. | β | 2022 | β |
| Massively parallel enrichment of low-frequency alleles enables duplex sequencing at low depth. | Gydush G et al. | β | 2022 | β |
| Multiplex technologies for the assessment of minimal residual disease and low-level mutation detection in leukaemia: mass spectrometry versus next-generation sequencing. | Shanmuganathan N et al. | β | 2022 | β |
| OPUSeq simplifies detection of low-frequency DNA variants and uncovers fragmentase-associated artifacts. | Alekseenko A et al. | β | 2022 | β |
| Potential clinical utility of liquid biopsies in ovarian cancer. | Zhu JW et al. | β | 2022 | β |
| Uterine lavage identifies cancer mutations and increased <i>TP53</i> somatic mutation burden in individuals with ovarian cancer. | Ghezelayagh TS et al. | β | 2022 | β |
| Commercial ctDNA Assays for Minimal Residual Disease Detection of Solid Tumors. | Chen K et al. | β | 2021 | β |
| Detection and Diagnostic Utilization of Cellular and Cell-Free Tumor DNA. | Dudley JC et al. | β | 2021 | β |
| Detection of low-frequency DNA variants by targeted sequencing of the Watson and Crick strands. | Cohen JD et al. | β | 2021 | β |
| Identifying the tissues-of-origin of circulating cell-free DNAs is a promising way in noninvasive diagnostics. | Peng X et al. | β | 2021 | β |
| Increased yields of duplex sequencing data by a series of quality control tools. | Povysil G et al. | β | 2021 | β |
| PolyG-DS: An ultrasensitive polyguanine tract-profiling method to detect clonal expansions and trace cell lineage. | Zhang Y et al. | β | 2021 | β |
| Prognostic and therapeutic implications of measurable residual disease in acute myeloid leukemia. | Aitken MJL et al. | β | 2021 | β |
| A novel virtual barcode strategy for accurate panel-wide variant calling in circulating tumor DNA. | Wu L et al. | β | 2020 | β |
| Family reunion via error correction: an efficient analysis of duplex sequencing data. | Stoler N et al. | β | 2020 | β |
| Multiplex accurate sensitive quantitation (MASQ) with application to minimal residual disease in acute myeloid leukemia. | Moffitt AB et al. | β | 2020 | β |
| Novel Illumina-based next generation sequencing approach with one-round amplification provides early and reliable detection of BCR-ABL1 kinase domain mutations in chronic myeloid leukemia. | Romzova M et al. | β | 2020 | β |
| RETrace: simultaneous retrospective lineage tracing and methylation profiling of single cells. | Wei CJ et al. | β | 2020 | β |
| Sensitive Identification of Bacterial DNA in Clinical Specimens by Broad-Range 16S rRNA Gene Enrichment. | Rassoulian Barrett S et al. | β | 2020 | β |
| The minimal that kills: Why defining and targeting measurable residual disease is the "Sine Qua Non" for further progress in management of acute myeloid leukemia. | Bewersdorf JP et al. | β | 2020 | β |
| Tumor DNA as a Cancer Biomarker through the Lens of Colorectal Neoplasia. | Cohen JD et al. | β | 2020 | β |
| Ultra-accurate Duplex Sequencing for the assessment of pretreatment ABL1 kinase domain mutations in Ph+ ALL. | Short NJ et al. | β | 2020 | β |
| Ultraconserved element bait set for trypanosomatida target enrichment and phylogenetics. | Kieran TJ | β | 2020 | β |
| Adaptive mutability of colorectal cancers in response to targeted therapies. | Russo M et al. | β | 2019 | β |
| A high-resolution landscape of mutations in the <i>BCL6</i> super-enhancer in normal human B cells. | Shen JC et al. | β | 2019 | β |
| Bioinformatics Analysis for Circulating Cell-Free DNA in Cancer. | Huang CC et al. | β | 2019 | β |
| Detection of Low-Frequency Mutations and Identification of Heat-Induced Artifactual Mutations Using Duplex Sequencing. | Ahn EH et al. | β | 2019 | β |
| Effect of low-level BCR-ABL1 kinase domain mutations identified by next-generation sequencing in patients with chronic myeloid leukaemia: a population-based study. | Kizilors A et al. | β | 2019 | β |
| Emerging translational science discoveries, clonal approaches, and treatment trends in chronic myeloproliferative neoplasms. | Mughal TI et al. | β | 2019 | β |
| Extensive subclonal mutational diversity in human colorectal cancer and its significance. | Loeb LA et al. | β | 2019 | β |
| FERMI: A Novel Method for Sensitive Detection of Rare Mutations in Somatic Tissue. | Liggett LA et al. | β | 2019 | β |
| High efficiency error suppression for accurate detection of low-frequency variants. | Wang TT et al. | β | 2019 | β |
| Immortalization of Different Breast Epithelial Cell Types Results in Distinct Mitochondrial Mutagenesis. | Kwon S et al. | β | 2019 | β |
| Isolation and Retrieval of Extracellular Vesicles for Liquid Biopsy of Malignant Ground-Glass Opacity. | Mao W et al. | β | 2019 | β |
| New approaches to molecular monitoring in CML (and other diseases). | Radich J et al. | β | 2019 | β |
| Quantitative detection of ALK fusion breakpoints in plasma cell-free DNA from patients with non-small cell lung cancer using PCR-based target sequencing with a tiling primer set and two-step mapping/alignment. | Kunimasa K et al. | β | 2019 | β |
| Targeted Single Primer Enrichment Sequencing with Single End Duplex-UMI. | Peng Q et al. | β | 2019 | β |
| Ultrasensitive Detection of Circulating Tumor DNA in Lymphoma via Targeted Hybridization Capture and Deep Sequencing of Barcoded Libraries. | Alcaide M et al. | β | 2019 | β |
| Ultra-Sensitive TP53 Sequencing for Cancer Detection Reveals Progressive Clonal Selection in Normal Tissue over a Century of Human Lifespan. | Salk JJ et al. | β | 2019 | β |
| Unexpectedly High Subclonal Mutational Diversity in Human Colorectal Cancer and Its Significance | Loeb LA et al. | β | 2019 | β |
| An ultrafast one-step assay for the visual detection of RNA virus. | Ma C et al. | β | 2018 | β |
| Detecting Rare Mutations and DNA Damage with Sequencing-Based Methods. | Sloan DB et al. | β | 2018 | β |
| Liquid biopsy and its role in an advanced clinical trial for lung cancer. | Johann DJ et al. | β | 2018 | β |
| Molecular Profiling of Liquid Biopsy Samples for Precision Medicine. | Campos CDM et al. | β | 2018 | β |
| Performance evaluation method for read mapping tool in clinical panel sequencing. | Lee H et al. | β | 2018 | β |
| Rapid and highly-specific generation of targeted DNA sequencing libraries enabled by linking capture probes with universal primers. | Pel J et al. | β | 2018 | β |
| Single-Molecule Sequencing Reveals Patterns of Preexisting Drug Resistance That Suggest Treatment Strategies in Philadelphia-Positive Leukemias. | Schmitt MW et al. | β | 2018 | β |
| Targeted genome fragmentation with CRISPR/Cas9 enables fast and efficient enrichment of small genomic regions and ultra-accurate sequencing with low DNA input (CRISPR-DS). | Nachmanson D et al. | β | 2018 | β |
| Validation of a Plasma-Based Comprehensive Cancer Genotyping Assay Utilizing Orthogonal Tissue- and Plasma-Based Methodologies. | Odegaard JI et al. | β | 2018 | β |
| Accurate RNA consensus sequencing for high-fidelity detection of transcriptional mutagenesis-induced epimutations. | Reid-Bayliss KS et al. | β | 2017 | β |
| Advances in Circulating Tumor DNA Analysis. | Perakis S et al. | β | 2017 | β |
| Asymmetrical barcode adapter-assisted recovery of duplicate reads and error correction strategy to detect rare mutations in circulating tumor DNA. | Ahn J et al. | β | 2017 | β |
| dCATCH-Seq: improved sequencing of large continuous genomic targets with double-hybridization. | Zhang Y et al. | β | 2017 | β |
| Detection of Rare Mutations in CtDNA Using Next Generation Sequencing. | Lv X et al. | β | 2017 | β |
| Evolutionary dynamics and significance of multiple subclonal mutations in cancer. | Beckman RA et al. | β | 2017 | β |
| High-throughput sequencing for noninvasive disease detection in hematologic malignancies. | Scherer F et al. | β | 2017 | β |
| Homeostasis Back and Forth: An Ecoevolutionary Perspective of Cancer. | Basanta D et al. | β | 2017 | β |
| Intratumor Heterogeneity: Novel Approaches for Resolving Genomic Architecture and Clonal Evolution. | Gupta RG et al. | β | 2017 | β |
| Involvement of autophagy in the outcome of mitotic catastrophe. | Sorokina IV et al. | β | 2017 | β |
| MAGERI: Computational pipeline for molecular-barcoded targeted resequencing. | Shugay M et al. | β | 2017 | β |
| Methyltransferase-Directed Labeling of Biomolecules and its Applications. | Deen J et al. | β | 2017 | β |
| Single molecule counting and assessment of random molecular tagging errors with transposable giga-scale error-correcting barcodes. | Lau BT et al. | β | 2017 | β |
| Targeted error-suppressed quantification of circulating tumor DNA using semi-degenerate barcoded adapters and biotinylated baits. | Alcaide M et al. | β | 2017 | β |
| Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq. | Wang K et al. | β | 2017 | β |
| Breast Cancer Heterogeneity Examined by High-Sensitivity Quantification of PIK3CA, KRAS, HRAS, and BRAF Mutations in Normal Breast and Ductal Carcinomas. | Myers MB et al. | β | 2016 | β |
| Cancer: hunting rare somatic mutations. | Marx V | β | 2016 | β |
| Cell-free DNA (cfDNA): Clinical Significance and Utility in Cancer Shaped By Emerging Technologies. | Volik S et al. | β | 2016 | β |
| Clonal haematopoiesis harbouring AML-associated mutations is ubiquitous in healthy adults. | Young AL et al. | β | 2016 | β |
| Current developments in molecular monitoring in chronic myeloid leukemia. | Marum JE et al. | β | 2016 | β |
| Decreased Mitochondrial Mutagenesis during Transformation of Human Breast Stem Cells into Tumorigenic Cells. | Ahn EH et al. | β | 2016 | β |
| Estimating Exceptionally Rare Germline and Somatic Mutation Frequencies via Next Generation Sequencing. | Eboreime J et al. | β | 2016 | β |
| Genome-wide mapping and analysis of chromosome architecture. | Schmitt AD et al. | β | 2016 | β |
| Genome-wide quantification of rare somatic mutations in normal human tissues using massively parallel sequencing. | Hoang ML et al. | β | 2016 | β |
| Heterozygous colon cancer-associated mutations of SAMHD1 have functional significance. | Rentoft M et al. | β | 2016 | β |
| Integrated digital error suppression for improved detection of circulating tumor DNA. | Newman AM et al. | β | 2016 | β |
| Rates and mechanisms of bacterial mutagenesis from maximum-depth sequencing. | Jee J et al. | β | 2016 | β |
| Statistical modeling for sensitive detection of low-frequency single nucleotide variants. | Hao Y et al. | β | 2016 | β |
| Streamlined analysis of duplex sequencing data with Du Novo. | Stoler N et al. | β | 2016 | β |
| The influence of subclonal resistance mutations on targeted cancer therapy. | Schmitt MW et al. | β | 2016 | β |
| TP53 mutations in epithelial ovarian cancer. | Zhang Y et al. | β | 2016 | β |
| Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues. | Krimmel JD et al. | β | 2016 | β |
| Aberrant reduction of telomere repetitive sequences in plasma cell-free DNA for early breast cancer detection. | Wu X et al. | β | 2015 | β |
| High definition viral vaccine strain identity and stability testing using full-genome population data--The next generation of vaccine quality control. | HΓΆper D et al. | β | 2015 | β |
| Targeting tumor suppressor genes for cancer therapy. | Liu Y et al. | β | 2015 | β |