RESUMEN
Replication stress (RS) is a key trait of cancer cells, and a potential actionable target in cancer treatment. Accurate methods to measure RS in tumour samples are currently lacking. DNA fibre analysis has been used as a common technique to measure RS in cell lines. Here, we investigated DNA fibre analysis on fresh breast cancer specimens and correlated DNA replication kinetics to known RS markers and genomic alterations. Fresh, treatment-naïve primary breast cancer samples (n = 74) were subjected to ex vivo DNA fibre analysis to measure DNA replication kinetics. Tumour cell proliferation was confirmed by EdU incorporation and cytokeratin AE1/AE3 (CK) staining. The RS markers phospho-S33-RPA and γH2AX and the RS-inducing proto-oncogenes Cyclin E1 and c-Myc were analysed by immunohistochemistry. Copy number variations (CNVs) were assessed from genome-wide single nucleotide polymorphism (SNP) arrays. We found that the majority of proliferating (EdU-positive) cells in each sample were CK-positive and therefore considered to be tumour cells. DNA fibre lengths varied largely in most tumour samples. The median DNA fibre length showed a significant inverse correlation with pRPA expression (r = -0.29, p = 0.033) but was not correlated with Cyclin E1 or c-Myc expression and global CNVs in this study. Nuclear Cyclin E1 expression showed a positive correlation with pRPA levels (r = 0.481, p < 0.0001), while cytoplasmic Cyclin E1 expression exhibited an inverse association with pRPA expression (r = -0.353, p = 0.002) and a positive association with global CNVs (r = 0.318, p = 0.016). In conclusion, DNA fibre analysis performed with fresh primary breast cancer samples is feasible. Fibre lengths were associated with pRPA expression. Cyclin E1 expression was associated with pRPA and the percentage of CNVs. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Asunto(s)
Neoplasias de la Mama , Ciclina E , Replicación del ADN , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Femenino , Ciclina E/genética , Ciclina E/metabolismo , Replicación del ADN/genética , Polimorfismo de Nucleótido Simple , Proliferación Celular , Variaciones en el Número de Copia de ADN , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Anciano , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , AdultoRESUMEN
Methylation-based liquid biopsies show promises in detecting cancer using circulating cell-free DNA; however, current limitations impede clinical application. Most assays necessitate substantial DNA inputs, posing challenges. Additionally, underrepresented tumor DNA fragments may go undetected during exponential amplification steps of traditional sequencing methods. Here, we report linear amplification-based bisulfite sequencing (LABS), enabling linear amplification of bisulfite-treated DNA fragments in a genome-wide, unbiased fashion, detecting cancer abnormalities with sub-nanogram inputs. Applying LABS to 100 patient samples revealed cancer-specific patterns, copy number alterations, and enhanced cancer detection accuracy by identifying tissue-of-origin and immune cell composition.
Asunto(s)
Metilación de ADN , Neoplasias , Análisis de Secuencia de ADN , Sulfitos , Humanos , Neoplasias/genética , Análisis de Secuencia de ADN/métodos , Ácidos Nucleicos Libres de Células , Técnicas de Amplificación de Ácido Nucleico/métodos , Variaciones en el Número de Copia de ADN , ADN de Neoplasias/genética , ADN Tumoral Circulante/genéticaAsunto(s)
Neoplasias Colorrectales , ADN de Neoplasias , Detección Precoz del Cáncer , Heces , Humanos , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/genética , ADN de Neoplasias/sangre , ADN de Neoplasias/análisis , Detección Precoz del Cáncer/métodos , Heces/química , Sangre Oculta , Secuenciación de Nucleótidos de Alto Rendimiento , Sensibilidad y Especificidad , Lesiones Precancerosas/diagnóstico , Lesiones Precancerosas/genéticaAsunto(s)
Neoplasias Colorrectales , ADN de Neoplasias , Detección Precoz del Cáncer , Heces , Humanos , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/genética , ADN de Neoplasias/sangre , ADN de Neoplasias/análisis , Detección Precoz del Cáncer/métodos , Heces/química , Sangre Oculta , Secuenciación de Nucleótidos de Alto Rendimiento , Sensibilidad y EspecificidadRESUMEN
PURPOSE OF REVIEW: This review sought to define the emerging roles of urinary tumor DNA (utDNA) for diagnosis, monitoring, and treatment of bladder cancer. Building from early landmark studies the focus is on recent studies, highlighting how utDNA could aid personalized care. RECENT FINDINGS: Recent research underscores the potential for utDNA to be the premiere biomarker in bladder cancer due to the constant interface between urine and tumor. Many studies find utDNA to be more informative than other biomarkers in bladder cancer, especially in early stages of disease. Points of emphasis include superior sensitivity over traditional urine cytology, broad genomic and epigenetic insights, and the potential for non-invasive, real-time analysis of tumor biology. utDNA shows promise for improving all phases of bladder cancer care, paving the way for personalized treatment strategies. Building from current research, future comprehensive clinical trials will validate utDNA's clinical utility, potentially revolutionizing bladder cancer management.
Asunto(s)
Biomarcadores de Tumor , ADN de Neoplasias , Neoplasias de la Vejiga Urinaria , Humanos , Neoplasias de la Vejiga Urinaria/orina , Neoplasias de la Vejiga Urinaria/diagnóstico , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/terapia , Biomarcadores de Tumor/orina , Biomarcadores de Tumor/genética , ADN de Neoplasias/orina , ADN de Neoplasias/genética , Medicina de Precisión/métodosRESUMEN
It is widely postulated that the majority of pathologically elevated extracellular or cell-free DNA (cfDNA) in cancer originates from tumor cells; however, evidence has emerged regarding the significant contributions of other cells from the tumor microenvironment. Here, the effect of cfDNA originating from murine B16 melanoma cells and L929 fibroblasts on B16 cells was investigated. It was found that cfDNAL929 increased the viability and migration properties of B16 cells in vitro and their invasiveness in vivo. In contrast, cfDNAB16 exhibited a negative effect on B16 cells, reducing their viability and migration in vitro, which in vivo led to decreased tumor size and metastasis number. It was shown that cell treatment with both cfDNAs resulted in an increase in the expression of genes encoding DNases and the oncogenes Braf, Kras, and Myc. cfDNAL929-treated cells were shown to experience oxidative stress. Gene expression changes in the case of cfDNAB16 treatment are well correlated with the observed decrease in proliferation and migration of B16 cells. The obtained data may indicate the possible involvement of fibroblast DNA in the tumor microenvironment in tumor progression and, potentially, in the formation of new tumor foci due to the transformation of normal cells.
Asunto(s)
Movimiento Celular , Ácidos Nucleicos Libres de Células , Fibroblastos , Melanoma Experimental , Microambiente Tumoral , Animales , Ratones , Fibroblastos/metabolismo , Melanoma Experimental/patología , Melanoma Experimental/metabolismo , Melanoma Experimental/genética , Microambiente Tumoral/genética , Ácidos Nucleicos Libres de Células/genética , Línea Celular Tumoral , Proliferación Celular , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , ADN de Neoplasias/metabolismo , ADN de Neoplasias/genética , Supervivencia Celular/efectos de los fármacos , Estrés OxidativoRESUMEN
Personalized cancer care requires molecular characterization of neoplasms. While the research community accepts frozen tissues as the gold standard analyte for molecular assays, the source of tissue for testing in clinical cancer care comes almost universally from formalin-fixed, paraffin-embedded tissue (FFPE). As newer technologies emerge for DNA characterization that requires higher molecular weight DNA, it was necessary to compare the quality of DNA in terms of DNA length between FFPE and cryopreserved samples. We hypothesized that cryopreserved samples would yield higher quantity and superior quality DNA compared to FFPE samples. We analyzed DNA metrics by performing a head-to-head comparison between FFPE and cryopreserved samples from 38 human tumors representing various cancer types. DNA quantity and purity were measured by UV spectrophotometry, and DNA from cryopreserved tissue demonstrated a 4.2-fold increase in DNA yield per mg of tissue (p-value < 0.001). DNA quality was measured on a fragment microelectrophoresis analyzer, and again, DNA from cryopreserved tissue demonstrated a 223% increase in the DNA quality number and a 9-fold increase in DNA fragments > 40,000 bp (p-value < 0.0001). DNA from the cryopreserved tissues was superior to the DNA from FFPE samples in terms of DNA yield and quality.
Asunto(s)
Criopreservación , Neoplasias , Adhesión en Parafina , Humanos , Criopreservación/métodos , Adhesión en Parafina/métodos , Neoplasias/genética , Fijación del Tejido/métodos , ADN/análisis , Formaldehído , ADN de Neoplasias/análisisRESUMEN
Objectives: In this study, stool samples were evaluated for tumor mutation analysis via a targeted next generation sequencing (NGS) approach in a small patient cohort suffering from localized rectal cancer. Introduction: Colorectal cancer (CRC) causes the second highest cancer-related death rate worldwide. Thus, improvements in disease assessment and monitoring that may facilitate treatment allocation and allow organ-sparing "watch-and-wait" treatment strategies are highly relevant for a significant number of CRC patients. Methods: Stool-based results were compared with mutation profiles derived from liquid biopsies and the gold standard procedure of tumor biopsy from the same patients. A workflow was established that enables the detection of de-novo tumor mutations in stool samples of CRC patients via ultra-sensitive cell-free tumor DNA target enrichment. Results: Notably, only a 19% overall concordance was found in mutational profiles across the compared sample specimens of stool, tumor, and liquid biopsies. Conclusion: Based on these results, the analysis of stool and liquid biopsy samples can provide important additional information on tumor heterogeneity and potentially on the assessment of minimal residual disease and clonal tumor evolution.
Asunto(s)
Biomarcadores de Tumor , Heces , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación , Neoplasias del Recto , Humanos , Heces/química , Neoplasias del Recto/genética , Neoplasias del Recto/patología , Neoplasias del Recto/sangre , Biomarcadores de Tumor/genética , Biopsia Líquida/métodos , Femenino , Masculino , ADN Tumoral Circulante/genética , ADN Tumoral Circulante/sangre , Persona de Mediana Edad , Anciano , Análisis Mutacional de ADN , Heterogeneidad Genética , ADN de Neoplasias/sangre , ADN de Neoplasias/genéticaRESUMEN
BACKGROUND: High-throughput sequencing (HTS) has become the gold standard approach for variant analysis in cancer research. However, somatic variants may occur at low fractions due to contamination from normal cells or tumor heterogeneity; this poses a significant challenge for standard HTS analysis pipelines. The problem is exacerbated in scenarios with minimal tumor DNA, such as circulating tumor DNA in plasma. Assessing sensitivity and detection of HTS approaches in such cases is paramount, but time-consuming and expensive: specialized experimental protocols and a sufficient quantity of samples are required for processing and analysis. To overcome these limitations, we propose a new computational approach specifically designed for the generation of artificial datasets suitable for this task, simulating ultra-deep targeted sequencing data with low-fraction variants and demonstrating their effectiveness in benchmarking low-fraction variant calling. RESULTS: Our approach enables the generation of artificial raw reads that mimic real data without relying on pre-existing data by using NEAT, a fine-grained read simulator that generates artificial datasets using models learned from multiple different datasets. Then, it incorporates low-fraction variants to simulate somatic mutations in samples with minimal tumor DNA content. To prove the suitability of the created artificial datasets for low-fraction variant calling benchmarking, we used them as ground truth to evaluate the performance of widely-used variant calling algorithms: they allowed us to define tuned parameter values of major variant callers, considerably improving their detection of very low-fraction variants. CONCLUSIONS: Our findings highlight both the pivotal role of our approach in creating adequate artificial datasets with low tumor fraction, facilitating rapid prototyping and benchmarking of algorithms for such dataset type, as well as the important need of advancing low-fraction variant calling techniques.
Asunto(s)
Benchmarking , Secuenciación de Nucleótidos de Alto Rendimiento , Neoplasias , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Neoplasias/genética , Mutación , Algoritmos , ADN de Neoplasias/genética , Análisis de Secuencia de ADN/métodos , Biología Computacional/métodosRESUMEN
PURPOSE: The Adaptively Dosed ImmunoTherapy Trial (ADAPT-IT;NCT03122522) investigated adaptive ipilimumab discontinuation in melanoma based on early radiographic assessment. Initial findings indicated similar effectiveness compared with conventional nivolumab-ipilimumab (nivo-ipi). Exploratory biomarker analyses and final clinical results are now reported. PATIENTS AND METHODS: Patients with unresectable melanoma received two doses of nivo-ipi. Radiographic assessment at Week 6 informed continuation of ipilimumab before nivolumab maintenance. The primary endpoint was overall response rate at Week 12. Plasma was assayed for circulating tumor DNA and 10 cytokines using a multiplex immunoassay. Flow cytometry of peripheral blood mononuclear cells was performed with an 11-color panel. RESULTS: Among the treated patients, expansion of proliferating T-cell populations was observed in responders and nonresponders. Baseline IL6 levels were low in patients achieving an objective radiographic response (median 1.30 vs. 2.86 pg/mL; P = 0.025). High baseline IL6 levels were associated with short progression-free survival [PFS; HR = 1.24, 95% confidence interval (CI), 1.01-1.52; P = 0.041]. At Week 6, patients with response had lower average tumor variant allele fractions than nonresponders (median 0.000 vs. 0.019; P = 0.014). Greater increases in average variant allele fractions from baseline to Week 6 correlated with short PFS (HR = 1.11, 95% CI, 1.01-1.21; P = 0.023). Week 12 overall response rate was 47% (95% CI, 35%-59%) with a median follow-up of 34 months among survivors. Median PFS was 21 months (95% CI, 10-not reached); 76% of responses (95% CI, 64%-91%) persisted at 36 months. CONCLUSIONS: Adaptively dosed nivo-ipi responses are durable and resemble historical data for conventional nivo-ipi. Baseline IL6 and circulating tumor DNA changes during treatment warrant further study as biomarkers of nivo-ipi response.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Citocinas , Ipilimumab , Melanoma , Nivolumab , Humanos , Nivolumab/administración & dosificación , Ipilimumab/administración & dosificación , Masculino , Femenino , Persona de Mediana Edad , Melanoma/tratamiento farmacológico , Melanoma/patología , Melanoma/mortalidad , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Adulto , Citocinas/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Anciano de 80 o más Años , Biomarcadores de Tumor , ADN de Neoplasias , ADN Tumoral CirculanteRESUMEN
BACKGROUND: Microsatellite instability (MSI) indicates DNA mismatch repair deficiency in certain types of cancer, such as colorectal cancer. The current gold standard technique, PCR-capillary electrophoresis (CE), requires matching normal samples and specialized instrumentation. We developed VarTrace, a rapid and low-cost quantitative PCR (qPCR) assay, to evaluate MSI using solely the tumor sample DNA, obviating the requirement for matching normal samples. METHODS: One hundred and one formalin-fixed paraffin-embedded (FFPE) tumor samples were tested using VarTrace and compared with the Promega OncoMate assay utilizing PCR-CE. Tumor percentage limit of detection was evaluated on contrived samples derived from clinical high MSI (MSI-H) samples. Analytical sensitivity, specificity, limit of detection, and input requirements were assessed using synthetic commercial reference standards. RESULTS: VarTrace successfully analyzed all 101 clinical FFPE samples, demonstrating 100% sensitivity and 98% specificity compared to OncoMate. It detected MSI-H with 97% accuracy down to 10% tumor. Analytical studies using synthetic samples showed a limit of detection of 5% variant allele frequency and a limit of input of 0.5 ng. CONCLUSIONS: This study validates VarTrace as a swift, accurate, and economical assay for MSI detection in samples with low tumor percentages without the need for matching normal DNA. VarTrace's capacity for highly sensitive MSI analysis holds potential for enhancing the efficiency of clinical work flows and broadening the availability of this test.
Asunto(s)
Inestabilidad de Microsatélites , Humanos , Adhesión en Parafina , Neoplasias/genética , Neoplasias/diagnóstico , Reacción en Cadena de la Polimerasa Multiplex/métodos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/diagnóstico , Sensibilidad y Especificidad , Electroforesis Capilar/métodos , Formaldehído , ADN de Neoplasias/genética , Límite de Detección , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Extrachromosomal DNA (ecDNA) is a self-replicating circular DNA originating from the chromosomal genome and exists outside the chromosome. It contains specific gene sequences and non-coding regions that regulate transcription. Recent studies have demonstrated that ecDNA is present in various malignant tumors. Malignant tumor development and poor prognosis may depend on ecDNA's distinctive ring structure, which assists in amplifying oncogenes. During cell division, an uneven distribution of ecDNA significantly enhances tumor cells' heterogeneity, allowing tumor cells to adapt to changes in the tumor microenvironment and making them more resistant to treatments. The application of ecDNA as a cancer biomarker and therapeutic target holds great potential. This article examines the latest advancements in this area and discusses the potential clinical applications of ecDNA.
Asunto(s)
ADN Circular , Neoplasias , Humanos , Neoplasias/genética , ADN Circular/genética , Animales , ADN de Neoplasias/genética , Biomarcadores de Tumor/genética , Microambiente Tumoral/genéticaAsunto(s)
Bacterias , Neoplasias , Humanos , Bacterias/genética , ADN de Neoplasias , Neoplasias/diagnóstico , Neoplasias/genéticaRESUMEN
Detection of peritoneal dissemination (PD) in gastric cancer (GC) patients remains challenging. The feasibility of tumor-guided cell-free DNA (cfDNA) detection in prospectively collected peritoneal fluid (ascites and peritoneal lavage) was investigated and compared to conventional cytology in 28 patients. Besides conventional cytology, next generation sequencing was performed on primary tumor DNA and cell-free DNA from peritoneal fluid. Patients were retrospectively grouped into: a positive group (with PD) and a negative group (without PD). Detectable mutations were found in the primary tumor of 68% (n = 19). Sensitivity of PD detection by tumor-guided cfDNA analysis was 91%, compared to 64% by conventional cytology. Within the positive group (n = 11), tumor-guided cfDNA was detected in all patients with ascites samples (4/4, 100%) and in 86% (6/7) of the lavage samples, opposed to 4/4 (100%) patients with ascites and 43% (3/7) with lavage by conventional cytology. Within the negative group (n = 8), conventional cytology was negative for all samples. In two patients, tumor-guided cfDNA was detected in peritoneal lavage fluid. Interestingly, these 2 patients developed PD within 6 months, suggesting a prognostic value of tumor-guided cfDNA detection. This study showed that tumor-guided cfDNA detection in peritoneal fluids of GC patients is feasible and superior to conventional cytology in detecting PD.
Asunto(s)
Líquido Ascítico , Ácidos Nucleicos Libres de Células , Neoplasias Peritoneales , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Neoplasias Gástricas/diagnóstico , Neoplasias Peritoneales/secundario , Neoplasias Peritoneales/genética , Neoplasias Peritoneales/diagnóstico , Femenino , Líquido Ascítico/patología , Líquido Ascítico/metabolismo , Masculino , Persona de Mediana Edad , Anciano , Ácidos Nucleicos Libres de Células/genética , Estudios Retrospectivos , ADN Tumoral Circulante/genética , Adulto , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Biomarcadores de Tumor/genética , Ascitis/genética , Ascitis/patología , Ascitis/diagnóstico , Mutación , Anciano de 80 o más Años , Lavado Peritoneal , ADN de Neoplasias/genética , ADN de Neoplasias/análisisRESUMEN
PURPOSE: Retinoblastoma (RB) is the most common intraocular tumor in pediatric age group. The role of genetics has been explored in predicting survival prognosis, but its role in predicting globe salvage remains largely unexplored. We hereby aim to isolate cell-free DNA (cfDNA) from aqueous humor (AH) in RB eyes and validate its use for genetic studies. METHODS: AH was obtained from 26 eyes undergoing enucleation (arm A) or intravitreal chemotherapy (arm B). Isolation of cfDNA was done using QIAamp ® Circulating Nucleic Acid kit, and the cfDNA was utilized for targeted sequencing of RB1 gene. RESULTS: We could isolate cfDNA in all eyes (72% unilateral and 28% bilateral) with a distribution peak between 140 and 160 bp and a mean concentration of 27.75 ng/µl for arm A and 14 ng/µl for arm B. Targeted sequencing done on four samples showed RB1 gene mutations, namely, inframe deletion (c. 78-80del, p.Pro29del), start-loss mutation (c.1A>T, p.Met1?), nonsense mutations (c.2236G>T, p.Glu746Ter), (c.1659T>A, p.Cys553Ter), and (c.2065C>T, p.Gln689Ter), and novel missense mutations (c.672C>A, p.Asp224Glu) and c.692C>T (p.Pro231Leu). Genetic profile of cfDNA extracted from AH and genomic DNA from the tumor tissue was comparable. CONCLUSION: Our study supports the previous reports that AH may be used as a source of tumor-derived cfDNA. This is the first report from South Asia on isolation and genetic analysis of cfDNA from AH of RB eyes and, therefore, a big step forward in paving the role of tumor genetics in RB. Further studies are required to elucidate concordance between the tumor and AH genetic profile.
Asunto(s)
Humor Acuoso , Neoplasias de la Retina , Retinoblastoma , Humanos , Retinoblastoma/genética , Retinoblastoma/diagnóstico , Neoplasias de la Retina/genética , Neoplasias de la Retina/diagnóstico , Humor Acuoso/metabolismo , Masculino , Femenino , Preescolar , Lactante , ADN de Neoplasias/genética , ADN de Neoplasias/análisis , Mutación , Enucleación del Ojo , Niño , Biomarcadores de Tumor/genética , India/epidemiología , Proteínas de Unión a Retinoblastoma/genética , Sur de Asia , Ubiquitina-Proteína LigasasRESUMEN
BACKGROUND: Hybridization capture-based targeted next generation sequencing (NGS) is gaining importance in routine cancer clinical practice. DNA library preparation is a fundamental step to produce high-quality sequencing data. Numerous unexpected, low variant allele frequency calls were observed in libraries using sonication fragmentation and enzymatic fragmentation. In this study, we investigated the characteristics of the artifact reads induced by sonication and enzymatic fragmentation. We also developed a bioinformatic algorithm to filter these sequencing errors. RESULTS: We used pairwise comparisons of somatic single nucleotide variants (SNVs) and insertions and deletions (indels) of the same tumor DNA samples prepared using both ultrasonic and enzymatic fragmentation protocols. Our analysis revealed that the number of artifact variants was significantly greater in the samples generated using enzymatic fragmentation than using sonication. Most of the artifacts derived from the sonication-treated libraries were chimeric artifact reads containing both cis- and trans-inverted repeat sequences of the genomic DNA. In contrast, chimeric artifact reads of endonuclease-treated libraries contained palindromic sequences with mismatched bases. Based on these distinctive features, we proposed a mechanistic hypothesis model, PDSM (pairing of partial single strands derived from a similar molecule), by which these sequencing errors derive from ultrasonication and enzymatic fragmentation library preparation. We developed a bioinformatic algorithm to generate a custom mutation "blacklist" in the BED region to reduce errors in downstream analyses. CONCLUSIONS: We first proposed a mechanistic hypothesis model (PDSM) of sequencing errors caused by specific structures of inverted repeat sequences and palindromic sequences in the natural genome. This new hypothesis predicts the existence of chimeric reads that could not be explained by previous models, and provides a new direction for further improving NGS analysis accuracy. A bioinformatic algorithm, ArtifactsFinder, was developed and used to reduce the sequencing errors in libraries produced using sonication and enzymatic fragmentation.