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Tissue sampling of biliary tract carcinomas (BTCs) for molecular characterization is challenging. The aim of this study was to investigate the possibility of identifying individual actionable mutations derived from bile cell-free DNA (cfDNA) using targeted deep sequencing. Ten BTC patients, four with gallbladder carcinomas and six with cholangiocarcinomas, were enrolled in the present study. Using targeted deep sequencing with a panel of 150 tumor-related genes, paired bile cfDNA and tumor DNA were analyzed for mutational variants individually and then compared. The present study, to the best of our knowledge, is the first to reveal that bile cfDNA is predominantly comprised of long DNA fragments, which is not the case for plasma cfDNA. Herein, paired bile cfDNA and tumors from ten BTC patients were examined using targeted deep sequencing. When comparing bile cfDNA and tumor DNA for single nucleotide variation (SNV)/insertion and deletion (Indel), the results using targeted deep sequencing revealed high sensitivity (94.7%) and specificity (99.9%). Additionally, the sensitivity of detecting a copy number variation (CNV) was 75.0%, with a specificity of 98.9%. When comparing two bile extraction methods, including percutaneous transhepatic cholangial drainage and operation, no significant difference in SNV/Indel or CNV detection sensitivity was noted. Moreover, when examining the tumor stage and incidence site, AJCC stage II and the distal bile duct both had significantly decreased CNV detection sensitivities. The present study revealed that targeted deep sequencing can reliably detect mutational variants within bile cfDNA obtained from BTC patients. These preliminary results may shed light on bile cfDNA as a promising liquid biopsy for BTC patients.

Background Targeted deep sequencing is increasingly used to detect low-allelic fraction variants; it is therefore essential that errors that constitute baseline noise and impose a practical limit on detection are characterized. In the present study, we systematically evaluate the extent to which errors are incurred during specific steps of the capture-based targeted sequencing process. Results We removed most sequencing artifacts by filtering out low-quality bases and then analyze the remaining background noise. By recognizing that plasma DNA is naturally fragmented to be of a size comparable to that of mono-nucleosomal DNA, we were able to identify and characterize errors that are specifically associated with acoustic shearing. Two-thirds of C:G > A:T errors and one quarter of C:G > G:C errors were attributed to the oxidation of guanine during acoustic shearing, and this was further validated by comparative experiments conducted under different shearing conditions. The acoustic shearing step also causes A > G and A > T substitutions localized to the end bases of sheared DNA fragments, indicating a probable association of these errors with DNA breakage. Finally, the hybrid selection step contributes to one-third of the remaining C:G > A:T and one-fifth of the C > T errors. Conclusions The results of this study provide a comprehensive summary of various errors incurred during targeted deep sequencing, and their underlying causes. This information will be invaluable to drive technical improvements in this sequencing method, and may increase the future usage of targeted deep sequencing methods for low-allelic fraction variant detection.

BackgroundHepatocellular carcinoma (HCC) recurrently develops in cirrhotic liver containing a number of regenerative nodules (RNs). However, the biological tumorigenic potential of RNs is still unclear. To uncover the molecular bases of tumorigenesis in liver cirrhosis, we investigated the genetic aberrations in RNs of cirrhotic tissues using next-generation sequencing.MethodsWe isolated 205 RNs and 7 HCC tissues from the whole explanted livers of 10 randomly selected patients who had undergone living-donor liver transplantation. Whole-exome sequencing and additional targeted deep sequencing on 30 selected HCC-related genes were conducted to reveal the mutational landscape of RNs and HCCs.ResultsWhole-exome sequencing demonstrated that RNs frequently harbored relatively high-abundance genetic alterations, suggesting a clonal structure of each RN in cirrhotic liver. The mutation signature observed in RNs was similar to those determined in HCC, characterized by a predominance of C>T transitions, followed by T>C and C>A mutations. Targeted deep sequencing analyses of RNs identified nonsynonymous low-abundance mutations in various tumor-related genes, including TP53 and ARID1A. In contrast, TERT promoter mutations were not detected in any of the RNs examined. Consistently, TERT expression levels in RNs were comparable to those in normal livers, whereas every HCC tissue demonstrated an elevated level of TERT expression.ConclusionAnalyses of RNs constructing cirrhotic liver indicated that a variety of genetic aberrations accumulate in the cirrhotic liver before the development of clinically and histologically overt HCC. These aberrations in RNs could provide the basis of tumorigenesis in patients with liver cirrhosis.

Cell lines are indispensable models for modern biomedical research. A large part of their usefulness derives from the ability of a cell line to proliferate over multiple passages (often indefinitely), allowing multiple experiments to be performed. However, over time, cell line identity and purity can be compromised by human errors. Cross-contamination from other cell lines and complete misidentification are both possible. Routine cell line authentication is a necessary preventive measure and has become a requirement for many funding applications and publications. Short tandem repeat (STR) profiling is the most common method for cell line authentication and is usually carried out using standard polymerase chain reaction-capillary electrophoresis analysis (STR-CE). Here, we evaluated next-generation sequencing (NGS)-based STR profiling of human and mouse cell lines at 18 and 15 loci, respectively, in a high-throughput format. Using the Python program STRight, we demonstrate that NGS-based analysis (STR-NGS) is superior to standard STR-CE in terms of the ability to report the sequence context of repeat motifs, sensitivity and flexible multiplexing capability. STR-NGS is thus a valuable alternative for cell line authentication.

BackgroundLiquid biopsies, particularly those involving circulating tumor DNA (ctDNA), are rapidly emerging as a non-invasive alternative to tumor biopsies. However, clinical applications of ctDNA analysis in hepatocellular carcinoma (HCC) have not been fully elucidated.MethodsWe measured the amount of plasma-derived cell-free DNA (cfDNA) in HCC patients before (n = 100) and a few days after treatment (n = 87), including radiofrequency ablation, transarterial chemoembolization, and molecular-targeted agents (MTAs), and prospectively analyzed their associations with clinical parameters and prognosis. TERT promoter mutations in cfDNA were analyzed using droplet digital PCR. Furthermore, we performed a comprehensive mutational analysis of post-treatment cfDNA via targeted ultra-deep sequencing (22,000× coverage) in a panel of 275 cancer-related genes in selected patients.ResultsPlasma cfDNA levels increased significantly according to HCC clinical stage, and a high cfDNA level was independently associated with a poor prognosis. TERT promoter mutations were detected in 45% of all cases but were not associated with any clinical characteristics. cfDNA levels increased significantly a few days after treatment, and a greater increase in post-treatment cfDNA levels was associated with a greater therapeutic response to MTAs. The detection rate of TERT mutations increased to 57% using post-treatment cfDNA, suggesting that the ctDNA was enriched. Targeted ultra-deep sequencing using post-treatment cfDNA after administering lenvatinib successfully detected various gene mutations and obtained promising results in lenvatinib-responsive cases.ConclusionsPost-treatment cfDNA analysis may facilitate the construction of biomarkers for predicting MTA treatment effects.

Abstract Rationale Uninvolved normal-appearing airway epithelium has been shown to exhibit specific mutations characteristic of nearby non–small cell lung cancers (NSCLCs). Yet, its somatic mutational landscape in patients with early-stage NSCLC is unknown. Objectives To comprehensively survey the somatic mutational architecture of the normal airway epithelium in patients with early-stage NSCLC. Methods Multiregion normal airways, comprising tumor-adjacent small airways, tumor-distant large airways, nasal epithelium and uninvolved normal lung (collectively airway field), matched NSCLCs, and blood cells (n = 498) from 48 patients were interrogated for somatic single-nucleotide variants by deep-targeted DNA sequencing and for chromosomal allelic imbalance events by genome-wide genotype array profiling. Spatiotemporal relationships between the airway field and NSCLCs were assessed by phylogenetic analysis. Measurements and Main Results Genomic airway field carcinogenesis was observed in 25 cases (52%). The airway field epithelium exhibited a total of 269 somatic mutations in most patients (n = 36) including key drivers that were shared with the NSCLCs. Allele frequencies of these acquired variants were overall higher in NSCLCs. Integrative analysis of single-nucleotide variants and allelic imbalance events revealed driver genes with shared “two-hit” alterations in the airway field (e.g., TP53, KRAS, KEAP1, STK11, and CDKN2A) and those with single hits progressing to two in the NSCLCs (e.g., PIK3CA and NOTCH1). Conclusions Tumor-adjacent and tumor-distant normal-appearing airway epithelia exhibit somatic driver alterations that undergo selection-driven clonal expansion in NSCLC. These events offer spatiotemporal insights into the development of NSCLC and, thus, potential targets for early treatment.

Members of the genus Populus L. play an important role in the formation of forests in the northern hemisphere and are used in urban landscaping and timber production. Populus species of closely related sections show extensive hybridization. Therefore, the systematics of the genus is rather complicated, especially for poplars of hybrid origin. We aimed to assess the efficiency of application of the sex-determining region (SDR) in addition to the nuclear and chloroplast genome loci traditionally used in phylogenetic studies of poplars to investigate relationships in sections Aigeiros Duby and Tacamahaca Spach. Targeted deep sequencing of NTS 5S rDNA, ITS, DSH 2 , DSH 5 , DSH 8 , DSH 12 , DSH 29 , 6 , 15 , 16 , X18 , trnG-psbK-psbI , rps2-rpoC2 , rpoC2-rpoC1 , as well as SDR and ARR17 gene was performed for 379 poplars. The SDR and ARR17 gene together with traditionally used multicopy and single-copy loci of nuclear and chloroplast DNA allowed us to obtain a clustering that is most consistent with poplar systematics based on morphological data and to shed light on several controversial hypotheses about the origin of the studied taxa (for example, the inexpediency of separating P. koreana , P. maximowiczii , and P. suaveolens into different species). We present a scheme of relationships between species and hybrids of sections Aigeiros and Tacamahaca based on molecular genetic, morphological, and geographical data. The geographical proximity of species and, therefore, the possibility of hybridization between them appear to be more important than the affiliation of species to the same section. We speculate that sections Aigeiros and Tacamahaca are distinguished primarily on an ecological principle (plain and mountain poplars) rather than on a genetic basis. Joint analysis of sequencing data for the SDR and chloroplast genome loci allowed us to determine the ancestors of P. × petrovskoe – P. laurifolia (female tree) × P. × canadensis (male tree), and P. × rasumovskoe – P. nigra (female tree) × P. suaveolens (male tree). Thus, the efficiency of using the SDR for the study of poplars of sections Aigeiros and Tacamahaca and the prospects of its use for the investigation of species of the genus Populus were shown.

Aims To investigate the key factors influencing glioma progression and the emergence of treatment resistance by examining the intrinsic connection between mutations in DNA damage and repair‐related genes and the development of chemoresistance in gliomas. Methods We conducted a comprehensive analysis of deep‐targeted gene sequencing data from 228 glioma samples. This involved identifying differentially mutated genes across various glioma grades, assessing their functions, and employing I‐TASSER for homology modeling. We elucidated the functional changes induced by high‐frequency site mutations in these genes and investigated their impact on glioma progression. Results The analysis of sequencing mutation results of deep targeted genes in integration revealed that ARID1A gene mutation occurs frequently in glioblastoma and alteration of ARID1A could affect the tolerance of glioma cells to temozolomide treatment. The deletion of proline at position 16 in the ARID1A protein affected the stability of binding of the SWI/SNF core subunit BRG1, which in turn affected the stability of the SWI/SNF complex and led to altered histone modifications in the CDKN1A promoter region, thereby affecting the biological activity of glioma cells, as inferred from modeling and protein interaction analysis. Conclusion The ARID1A gene is a critical predictive biomarker for glioma. Mutations at the ARID1A locus alter the stability of the SWI/SNF complex, leading to changes in transcriptional regulation in glioma cells. This contributes to an increased malignant phenotype of GBM and plays a pivotal role in mediating chemoresistance. Deep sequencing targeting DNA damage repair pathways identifies ARID1A as a prognostic biomarker for gliomas and its mutation alters SWI/SNF stability increasing the malignant phenotype of GBM and mediating chemoresistance.

Background Recently, a next-generation sequencing (NGS)-based method has been used for the successful detection of circulating tumor DNA (ctDNA) in various cancer types. Thus, the use of NGS on liquid biopsies will improve cancer diagnosis and prognosis. However, the low-allelic fraction of ctDNA poses a challenge for the sensitive and specific detection of tumor variants in cell-free DNA (cfDNA). To distinguish true variants from false positives, the characteristics of errors that occur during sample preparation and sequencing need to be elucidated. Methods We generated capture-based targeted deep sequencing data from plasma cfDNA and peripheral blood leucocyte (PBL) gDNA to profile background errors. To reveal cfDNA-associated DNA lesions, background error profiles from two sample types were compared in each nucleotide substitution class. Results In this study, we determined the prevalence of single nucleotide substitutions in cfDNA sequencing data to identify DNA damage preferentially associated with cfDNA. On comparing sequencing errors between cfDNA and cellular genomic DNA (gDNA), we observed that the total substitution error rates in cfDNA were significantly higher than those in gDNA. When the substitution errors were divided into 12 substitution error classes, C:G>T:A substitution errors constituted the largest difference between cfDNA and gDNA samples. When the substitution error rates were estimated based on the location of DNA-fragment substitutions, the differences in error rates of most substitution classes between cfDNA and gDNA samples were observed only at the ends of the DNA fragments. In contrast, C:G>T:A substitution errors in the cfDNA samples were not particularly associated with DNA-fragment ends. All observations were verified in an independent dataset. Conclusions Our data suggested that cytosine deamination increased in cfDNA compared to that in cellular gDNA. Such an observation might be due to the attenuation of DNA damage repair before the release of cfDNA and/or the accumulation of cytosine deamination after it. These findings can contribute to a better understanding of cfDNA-associated DNA damage, which will enable the accurate analysis of somatic variants present in cfDNA at an extremely low frequency.

Hepatocellular carcinoma (HCC) is the most common malignancy of the liver. Genomic analysis is conducted to identify genetic alterations in driver genes which are all druggable targets for cancer therapy. In the present study, we performed an exome sequencing of 45 driver genes in 100 paired samples from HCC patients including tumors and matched adjacent normal tissues using Illumina HiSeq 2000 platform. Non-synonymous mutations were ascertained using the iPLEX MassARRAY system and Sanger sequencing. Clinicopathological relevance with genetic variations was assessed using SPSS software. The prognostic analyses of patients with gene mutation status were summarized using Kaplan-Meier curves. Sixty-one non-synonymous somatic mutations were identified in 43% of the HCC patients. The most frequent mutations were: TP53 (20%), RET (6%), PLCE1 (5%), PTEN (4%) and VEGFR2 (3%). Patients with mutations in TP53 had a lower overall survival (OS) (P=0.002) than those without mutations. Recurrent mutations in the Ret proto-oncogene (RET) were associated with poor outcomes for both disease-free survival (DFS) (P=0.028) and OS (P=0.001) in HCC patients. The mutational status of sorafenib-targeted genes were associated with decreased DFS (P=0.039), and decreased OS (P=0.15) without statistical significance. Mutual exclusion of TP53 and RET mutations were observed in the present study. In conclusion, patients with TP53 mutations, RET mutations and sorafenib-targeted gene mutations were demonstrated to be associated with poor HCC prognosis, which suggests that both TP53 and RET may serve as biomarkers of prognostic evaluation and targeted therapy in HCC.