Combination Of Next-generation Sequencing Research Articles

The combination of next generation sequencing and technological devices allows a precision medicine approach in congenital hyperinsulinism: the case of a pregnant mother and the child she gave birth.

The combination of next generation sequencing and technological devices allows a precision medicine approach in congenital hyperinsulinism: the case of a pregnant mother and the child she gave birth.

Evaluation of 16S-Based Metagenomic NGS as Diagnostic Tool in Different Types of Culture-Negative Infections.

Bacterial infections pose significant global health challenges, often underestimated due to difficulties in accurate diagnosis, especially when culture-based diagnostics fail. This study assesses the effectiveness of 16S-based metagenomic next generation sequencing (NGS) for identifying pathogens in culture-negative clinical samples across various medical settings. Overall, 48% of samples were collected from orthopedics, 15% from neurosurgery, and 12% in cardiac surgery, among others. The detection rate of monomicrobial infections was 68.6%, and 5.7% for polymicrobial infections. In addition, NGS detected bacteria in all samples from the lungs, head and neck, and eye specimens. Cutibacterium acnes (11%, 12/105) was the most frequent microorganism, followed by Staphylococcus epidermidis (10.4%, 11/105), and Staphylococcus aureus (9.5%, 10/105). In conclusion, 16S-targeted metagenomic sequencing enhances pathogen detection capabilities, particularly in instances where traditional cultures fail. By the combination of NGS and bacterial cultures, microbiologists might provide a more accurate diagnosis, guiding more effective treatments and potentially reducing healthcare costs associated with empirical treatments.

Full-Length Transcriptome Sequencing and RNA-Seq Analysis Offer Insights into Terpenoid Biosynthesis in Blumea balsamifera (L.) DC.

Blumea balsamifera (L.) DC., an important economic and medicinal herb, has a long history of being used as a traditional Chinese medicine. Its leaves have always been used as a raw material for the extraction of essential oils, comprising large amounts of terpenoids, which have good therapeutic effects on many diseases, such as eczema, bacterial infection, and hypertension. However, the genetic basis of terpenoid biosynthesis in this plant is virtually unknown on account of the lack of genomic data. Here, a combination of next-generation sequencing (NGS) and full-length transcriptome sequencing was applied to identify genes involved in terpenoid biosynthesis at five developmental stages. Then, the main components of essential oils in B. balsamifera were identified using GC-MS. Overall, 16 monoterpenoids and 20 sesquiterpenoids were identified and 333,860 CCS reads were generated, yielding 65,045 non-redundant transcripts. Among these highly accurate transcripts, 59,958 (92.18%) transcripts were successfully annotated using NR, eggNOG, Swissprot, KEGG, KOG, COG, Pfam, and GO databases. Finally, a total of 56 differently expressed genes (DEGs) involved in terpenoid biosynthesis were identified, including 38 terpenoid backbone genes and 18 TPSs, which provide a significant amount of genetic information for B. balsamifera. These results build a basis for resource protection, molecular breeding, and the metabolic engineering of this plant.

Abstract A036: The genetic landscape of head and neck cancer using brush biopsy

Abstract Oral potentially malignant disorders (OPMDs) are characterized by the presence of dysplasia and DNA copy number aberrations (CNAs) and subsequently progress to oral squamous cell carcinoma (OSCC). OSCC has a 5-year survival rate of only about 50% and varies greatly depending on site and stage. The combination of next-generation sequencing and advanced computational data analysis approaches has revolutionized our understanding of the genomic underpinnings of cancer development and progression. This can reveal individual targetable alterations, mutational load, complex mutation signatures, and tumor-specific antigens, which might inform the utilization of targeted therapies and early diagnosis. Oral brush biopsy represents an attractive non-invasive advantage in the characterization of premalignant and malignant oral lesions. Still, a sufficient number of abnormal cells is mandatory to diagnose. The current study tested the possibility of recreating the genomic landscape of oral cancer using brush biopsy. Materials and methods: As a pilot study to address this question, we analyzed matched tissue and brush biopsy of a patient with synchronous OPMD and OSCC. Brush and tissue biopsy was taken from normal epithelium, OPMD, and OSCC. We scrutinized the evolution and heterogeneity of these three brush/tissue biopsy pairs. The techniques used include whole exome sequencing (WES), mutational signature analysis to evaluate smoking and other aberrant signatures, copy number analysis (using ASCAT), and subclonal architecture reconstruction (using DPclust). Results: To analyze intra-tumoral heterogeneity (ITH), ASCAT and DPclust were used to call copy number aberrations (CNA) and cluster somatic SNVs based on cancer cell fraction (CCF), respectively. We observed a large number of shared SNVs between the OPMD and OSCC lesions suggesting a common evolutionary origin. After adjusting the ploidy and purity based on DPClust results, ASCAT profiles revealed differences in aberrant tumor cell fraction, gains and loss of heterozygosity, and copy number neutral events between the samples. It showed almost similar profiles between the respective lesions' brush and tissue samples, suggesting that brush biopsy can recapitulate the genomic landscape. The OPMD and OSCC lesions showed loss of heterozygosity in chromosome 9 and chromosome 17, which play a potential role in tumorigenesis with loss of function of P53. We inferred the subclonal architecture of each sample, illuminating the clonal relationships between samples and distinct lineages of precursor clones developing independently. Conclusion: Collectively, these results emphasize the possibility of using brush biopsy to reconstruct the genomic characterization of OPMD and OSCC show prognostic potential for diagnosis and intervention strategies, and accelerate progress in precision cancer care. Citation Format: Evit John, Tom Lesluyes, Xiao Zhao, Peter Van Loo. The genetic landscape of head and neck cancer using brush biopsy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A036.

Characterization of Robertsonian and Reciprocal Translocations in Cattle through NGS.

This study presents a novel approach that combines next-generation sequencing (NGS) and cytogenetic technologies for identifying chromosomes involved in chromosomal anomalies. This research focuses on a chromosome anomaly discovered in male Alpine Grey cattle, as well as two previously reported cases of reciprocal translocations (rcps), namely rcp(9;11) and rcp(4;7). Abnormal chromosomes from Alpine Grey cattle were microdissected from conventional preparations, and the amplified products were sequenced using NGS. The sequencing reads were then mapped to the reference genome, and the leverage effect was calculated to identify abnormal reads/Mb values. The result revealed the presence of rob(26;29), which was further confirmed through traditional cytogenetic analyses such as Giemsa staining, CBA-banding, RBA-banding, and FISH techniques. Furthermore, the feasibility of this approach on preserved metaphases was demonstrated through analysis of old slides from previously characterized cases. The study highlights the challenges involved in identifying and characterizing chromosomal aberrations in bovine species and offers a potential solution for analyzing historical anomalies when fresh blood material is unavailable. The combination of NGS and cytogenetic techniques provides a cost-effective and reliable approach for characterizing chromosomal anomalies in various species, including those identified before the availability of modern banding technologies and FISH mapping using specific molecular markers.

Donor white blood cell differential is the single largest determinant of whole blood gene expression patterns

It has become widely accepted that sample cellular composition is a significant determinant of the gene expression patterns observed in any transcriptomic experiment performed with bulk tissue. Despite this, many investigations currently performed with whole blood do not experimentally account for possible inter-specimen differences in cellularity, and often assume that any observed gene expression differences are a result of true differences in nuclear transcription. In order to determine how confounding of an assumption this may be, in this study, we recruited a large cohort of human donors (n = 138) and used a combination of next generation sequencing and flow cytometry to quantify and compare the underlying contributions of variance in leukocyte counts versus variance in other biological factors to overall variance in whole blood transcript levels. Our results suggest that the combination of donor neutrophil and lymphocyte counts alone are the primary determinants of whole blood transcript levels for up to 75% of the protein-coding genes expressed in peripheral circulation, whereas the other factors such as age, sex, race, ethnicity, and common disease states have comparatively minimal influence. Broadly, this infers that a majority of gene expression differences observed in experiments performed with whole blood are driven by latent differences in leukocyte counts, and that cell count heterogeneity must be accounted for to meaningfully biologically interpret the results.

Abstract PO-015: The genetic landscape of head and neck cancer using brush biopsy

Abstract Oral potentially malignant disorders (OPMDs) are characterized by the presence of dysplasia and DNA copy number aberrations (CNAs) and subsequently progress to oral squamous cell carcinoma (OSCC). OSCC has a 5-year survival rate of only about 50% and varies greatly depending on site and stage. The combination of next-generation sequencing and advanced computational data analysis approaches has revolutionized our understanding of the genomic underpinnings of cancer development and progression. This can reveal individual targetable alterations, mutational load, complex mutation signatures, and tumor-specific antigens, which might inform the utilization of targeted therapies and early diagnosis. Oral brush biopsy represents an attractive non-invasive advantage in the characterization of premalignant and malignant oral lesions. Still, a sufficient number of abnormal cells is mandatory to diagnose. The current study tested the possibility of recreating the genomic landscape of oral cancer using brush biopsy. Materials and methods: As a pilot study to address this question, we analyzed matched tissue and brush biopsy of a patient with synchronous OPMD and OSCC. Brush and tissue biopsy was taken from normal epithelium, OPMD, and OSCC. We scrutinized the evolution and heterogeneity of these three brush/tissue biopsy pairs. The techniques used include whole exome sequencing (WES), mutational signature analysis to evaluate smoking and other aberrant signatures, copy number analysis (using ASCAT), and subclonal architecture reconstruction (using DPclust). Results: To analyze intra-tumoral heterogeneity (ITH), ASCAT and DPclust were used to call copy number aberrations (CNA) and cluster somatic SNVs based on cancer cell fraction (CCF), respectively. We observed a large number of shared SNVs between the OPMD and OSCC lesions suggesting a common evolutionary origin. After adjusting the ploidy and purity based on DPClust results, ASCAT profiles revealed differences in aberrant tumor cell fraction, gains and loss of heterozygosity, and copy number neutral events between the samples. It showed almost similar profiles between the respective lesions' brush and tissue samples, suggesting that brush biopsy can recapitulate the genomic landscape. The OPMD and OSCC lesions showed loss of heterozygosity in chromosome 9 and chromosome 17, which play a potential role in tumorigenesis with loss of function of P53. We inferred the subclonal architecture of each sample, illuminating the clonal relationships between samples and distinct lineages of precursor clones developing independently. Conclusion: Collectively, these results emphasize the possibility of using brush biopsy to reconstruct the genomic characterization of OPMD and OSCC and show prognostic potential for diagnosis and intervention strategies and accelerate progress in precision cancer care. Citation Format: Evit John, Tom Lesluyes, Toby Baker, Xiao Zhao, Peter Van Loo. The genetic landscape of head and neck cancer using brush biopsy [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-015.

Illumina RNA and SMRT Sequencing Reveals the Mechanism of Uptake and Transformation of Selenium Nanoparticles in Soybean Seedlings

Selenium (Se) is an essential element for mammals, and its deficiency in the diet is a global problem. Agronomic biofortification through exogenous Se provides a valuable strategy to enhance human Se intake. Selenium nanoparticles (SeNPs) have been regarded to be higher bioavailability and less toxicity in comparison with selenite and selenate. Still, little has been known about the mechanism of their metabolism in plants. Soybean (Glycine max L.) can enrich Se, providing an ideal carrier for Se biofortification. In this study, soybean sprouts were treated with SeNPs, and a combination of next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing was applied to clarify the underlying molecular mechanism of SeNPs metabolism. A total of 74,662 nonredundant transcripts were obtained, and 2109 transcription factors, 9687 alternative splice events, and 3309 long non-coding RNAs (lncRNAs) were predicted, respectively. KEGG enrichment analysis of the DEGs revealed that metabolic pathways, biosynthesis of secondary metabolites, and peroxisome were most enriched both in roots and leaves after exposure to SeNPs. A total of 117 transcripts were identified to be putatively involved in SeNPs transport and biotransformation in soybean. The top six hub genes and their closely coexpressed Se metabolism-related genes, such as adenylylsulfate reductase (APR3), methionine-tRNA ligase (SYM), and chloroplastic Nifs-like cysteine desulfurases (CNIF1), were screened by WGCNA and identified to play crucial roles in SeNPs accumulation and tolerance in soybean. Finally, a putative metabolism pathway of SeNPs in soybean was proposed. These findings have provided a theoretical foundation for future elucidation of the mechanism of SeNPs metabolism in plants.

Response of soil microbial compositional and functional heterogeneity to grazing exclusion in alpine shrub and meadows in the Qinghai-Tibet Plateau.

Soil microorganisms found in shrub-meadow ecosystems are highly heterogeneous and extremely sensitive to grazing, but changes in microbial compositional and functional heterogeneity during grazing exclusion (GE) have been largely overlooked compared to community diversity. We collected soil samples from heavily grazed plots (6.0 sheep/ha) and GE plots (matrix and patch areas in both), and used a combination of next-generation sequencing, vegetation features, and the associated soil property data to investigate the effect of GE on the composition and function of microbial communities (bacteria fungi, and archaea) in 0-10 cm soils. Regarding community composition, the proportions of species in bacteria, fungi, and archaea were 97.3, 2.3, and 0.4%, respectively. GE significantly affected the species diversity of fungi and archaea but not that of bacteria. GE decreased the heterogeneity of bacteria (2.9% in matrix and 6.2% in patch) and archaea (31.1% in matrix and 19.7% in patch) but increased that of fungi by 1.4% in patch. Regarding community function, enzyme diversity and heterogeneity were increased by 10.4 and 9.4%, respectively, in patch after 6 years of fencing, exemplifying a high level of microbial functional redundancy. The Kyoto Encyclopedia of Genes and Genome pathways-cell growth and death, translation, digestive system, and nucleotide metabolism-were functional biomarkers (linear discriminant analysis effect size method) in matrix-non-grazed plots, whereas lipid metabolism, xenobiotics biodegradation and metabolism, and metabolism of terpenoids and polyketides, cell motility, cancer: overview, endocrine system, and membrane transport were biomarkers in patch-non-grazed plots. Additionally, GE improved the capacity for fatty acid metabolism but decreased the abundance of methane-producing archaea by 42.9%. Redundancy analysis revealed that the factors that affected microbial composition the most were soil aggregates, soil moisture, and the number of plant species, whereas those that affected microbial function the most were soil available phosphorus, soil temperature, and shrub canopy diameter. Our results quantified soil microbial heterogeneity, emphasizing the different responses of the composition and function of bacteria, fungi, and archaea to GE in alpine shrubs and meadows.

Next-Generation Phage Display to Identify Peptide Ligands of Deubiquitinases.

Phage display (PD) is a powerful method and has been extensively used to generate monoclonal antibodies and identify epitopes, mimotopes, and protein interactions. More recently, the combination of next-generation sequencing (NGS) with PD (NGPD)has revolutionized the capabilities of the method by creating large data sets of sequences from affinity selection-based approaches (biopanning) otherwise challenging to obtain. NGPD can monitor motif enrichment, allow tracking of the selection process over consecutive rounds, and highlight unspecific binders. To tackle the wealth of data obtained, bioinformatics tools have been developed that allow for identifying specific binding sequences (binders) that can then be validated. Here, we provide a detailed account of the use of NGPD experiments to identify ubiquitin-specific protease peptide ligands.

Over two decades of research on the marine RNA virosphere.

RNA viruses (realm: Riboviria), including RNA phages and eukaryote-infecting RNA viruses, are essential components of marine ecosystems. A large number of marine RNA viruses have been discovered in the last two decades because of the rapid development of next-generation sequencing (NGS) technology. Indeed, the combination of NGS and state-of-the-art meta-omics methods (viromics, the study of all viruses in a specific environment) has led to a fundamental understanding of the taxonomy and genetic diversity of RNA viruses in the sea, suggesting the complex ecological roles played by RNA viruses in this complex ecosystem. Furthermore, comparisons of viromes in the context of highly variable marine niches reveal the biogeographic patterns and ecological impact of marine RNA viruses, whose role in global ecology is becoming increasingly clearer. In this review, we summarize the characteristics of the global marine RNA virosphere and outline the taxonomic hierarchy of RNA viruses with a specific focus on their ancient evolutionary history. We also review the development of methodology and the major progress resulting from its applications in RNA viromics. The aim of this review is not only to provide an in-depth understanding of multifaceted aspects of marine RNA viruses, but to offer future perspectives on developing a better methodology for discovery, and exploring the evolutionary origin and major ecological significance of marine RNA virosphere.

Genotype Complements the Phenotype: Identification of the Pathogenicity of an LMNA Splice Variant by Nanopore Long-Read Sequencing in a Large DCM Family.

Dilated cardiomyopathy (DCM) is a common cause of heart failure (HF) and is of familial origin in 20–40% of cases. Genetic testing by next-generation sequencing (NGS) has yielded a definite diagnosis in many cases; however, some remain elusive. In this study, we used a combination of NGS, human-induced pluripotent-stem-cell-derived cardiomyocytes (iPSC-CMs) and nanopore long-read sequencing to identify the causal variant in a multi-generational pedigree of DCM. A four-generation family with familial DCM was investigated. Next-generation sequencing (NGS) was performed on 22 family members. Skin biopsies from two affected family members were used to generate iPSCs, which were then differentiated into iPSC-CMs. Short-read RNA sequencing was used for the evaluation of the target gene expression, and long-read RNA nanopore sequencing was used to evaluate the relevance of the splice variants. The pedigree suggested a highly penetrant, autosomal dominant mode of inheritance. The phenotype of the family was suggestive of laminopathy, but previous genetic testing using both Sanger and panel sequencing only yielded conflicting evidence for LMNA p.R644C (rs142000963), which was not fully segregated. By re-sequencing four additional affected family members, further non-coding LMNA variants could be detected: rs149339264, rs199686967, rs201379016, and rs794728589. To explore the roles of these variants, iPSC-CMs were generated. RNA sequencing showed the LMNA expression levels to be significantly lower in the iPSC-CMs of the LMNA variant carriers. We demonstrated a dysregulated sarcomeric structure and altered calcium homeostasis in the iPSC-CMs of the LMNA variant carriers. Using targeted nanopore long-read sequencing, we revealed the biological significance of the variant c.356+1G>A, which generates a novel 5′ splice site in exon 1 of the cardiac isomer of LMNA, causing a nonsense mRNA product with almost complete RNA decay and haploinsufficiency. Using novel molecular analysis and nanopore technology, we demonstrated the pathogenesis of the rs794728589 (c.356+1G>A) splice variant in LMNA. This study highlights the importance of precise diagnostics in the clinical management and workup of cardiomyopathies.

The diagnostic value of next-generation sequencing technology in sepsis.

ObjectiveThis study aims to assess the clinical utility of next-generation sequencing (NGS) in sepsis diagnosis.MethodsA prospective study was conducted on patients with a high suspicion of sepsis by unknown pathogens from January 2017 to December 2021. Blood samples were taken from patients to perform NGS, blood culture (BC), leucocyte (WBC), procalcitonin (PCT), creatinine (CREA), Albumin (ALB) and C-reactive protein (CRP) tests.ResultsThe feedback time for BC was 3~5 days for bacteria and 5~7 days for fungi, while the turnover time for NGS was only 24 h. The clinical diagnosis was considered the “gold standard”. 83 patients passed our inclusion criteria and were separated into two groups by clinical diagnosis. 62 met the clinical diagnosis criteria for sepsis and 21 were non-sepsis. The data from the two groups were retrospectively compared and analyzed. Of 62 sepsis in 83 patients, 8(9.64%) were diagnosed by both BC and NGS, 51 (61.45%) by NGS only, 1(1.20%) by BC and 2 (2.41%) by conventional testing only; PCT, CREA, CRP levels and the detection rate of NGS and BC were higher in the sepsis group than in the non-sepsis group, while ALB levels were lower (p<0.05). The logistic regression results in our study revealed that NGS and ALB were independent prediction factors for sepsis (p<0.05), the area under the receiver operating characteristic curve (AUC), sensitivity and specificity of NGS for diagnosing sepsis was 0.857, 95.16% and 76.19%, while ALB was 0.728, 58.06%, 80.95%, respectively. The combination’s sensitivity, specificity and AUC of NGS and ALB were 93.55%, 85.71% and 0.935, greater than that of Albumin or NGS only (both p<0.05).ConclusionNGS can effectively and quickly identify pathogens, thereby emerges as a promising technology for sepsis diagnosis. Combination of NGS and ALB can be used for early screening and is more powerful than NGS or ALB only.

Accurate detection of Escherichia coli O157:H7 and Salmonella enterica serovar typhimurium based on the combination of next-generation sequencing and droplet digital PCR

Shiga-toxin producing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium are important foodborne pathogens which are threatened to human health seriously. In this study, we firstly investigated the application and accuracy of next-generation sequencing (NGS) technology combining with droplet digital PCR (NGS + ddPCR) for the quantification of pathogenic bacteria through amplification of 16S rRNA genes. Absolute quantification by ddPCR was then further developed to identify E. coli O157:H7 and S. Typhimurium targeting the single copy genes Z3276 and invA, respectively. Apple juice, milk and chicken were selected as corresponding represents of fruit, dairy and meat, to investigate the application of ddPCR in different food. The tested microbial communities were all successfully annotated by NGS at the level of species, and the accuracy of absolute abundance calculated by NGS + ddPCR relied on its initial population for each species. When detecting the genes Z3276 and invA simultaneously by dual channels using ddPCR, the detection limit of E. coli O157:H7 and S. Typhimurium in apple juice and milk was 2–3 log cfu/mL, while it was increased to 3–4 log cfu/g on chicken surface. Exist of background species or homologous strains did not affect the accuracy or detection limit of the targeting strain.

Identification and therapeutic evaluation of ALK rearrangements in non-small-cell lung cancer.

This study aimed to present a comprehensive assessment of anaplastic lymphoma kinase (ALK) rearrangements evaluated by DNA/RNA‐based next‐generation sequencing (NGS) and Ventana immunohistochemistry (IHC) in patients with non‐small‐cell lung cancer (NSCLC) and to evaluate the therapeutic outcomes of ALK tyrosine kinase inhibitor (TKI) treatment. We investigated ALK gene fusions in 14,894 patients with NSCLC using Ventana IHC and NGS, including 12,533 cases detected via DNA‐based NGS and 2,361 cases using RNA‐based NGS. The overall percentage agreement (OPA), positive percentage agreement (PPA), and negative percentage agreement (NPA) were calculated when comparing the results between NGS and IHC. The therapeutic responses to ALK‐TKIs were also evaluated. In total, 3.50% (439/12,533) of specimens were NGS ALK‐positive (NGS‐p) in the DNA‐based NGS cohort and 3.63% (455/12,533) were IHC ALK‐positive (IHC‐p). The OPA of NGS was 99.60%, whereas its PPA and NPA were 92.75 and 99.86%, respectively. In the adenocarcinoma (ADC) subcohort, the PPA was 95.69%. In the RNA‐based NGS cohort, 2.20% (52/2,361) of specimens were NGS‐p and 2.63% (62/2,361) were IHC‐p. The OPA of NGS was 99.49%; its PPA and NPA were 82.26 and 99.96%, respectively. Thirteen patients with discordant results received ALK‐TKI treatment. In the seven NGS‐p/IHC‐negative (IHC‐n) patients, the overall response rate (ORR) was 85.4% (6/7) and the disease control rate (DCR) was 100%. In the six NGS‐negative/IHC‐p patients, the ORR was 66.7% (4/6) and the DCR was 100%. In summary, a high concordance of ALK gene fusion detected via NGS and IHC was observed in this study. DNA‐based NGS had a higher OPA, PPA, and PPA in the ADC subcohort, whereas RNA‐based NGS had a higher NPA. Overall, the results suggest that the combination of NGS and IHC can improve the accuracy of ALK fusion detection; hence, a result determination algorithm for clinical detection of ALK gene fusion was also proposed.

Next-generation sequencing and targeted quantitative real-time polymerase chain reaction for detection of Akebiae Caulis in the traditional Chinese medical formula Longdan Xiegan Wan.

BackgroundAkebiae Caulis (Mu Tong) is commonly misused by Aristolochiae Manshuriensis Caulis (Guan Mutong) and Clematidis Armandii Caulis (Chuan Mutong), which are nephrotoxic and carcinogenic. However, in the Pharmacopoeia of the People’s Republic of China (2015 Edition), the method for determining Akebiae Caulis remains undefined.MethodsWe used DNA barcode-based next-generation sequencing (NGS) combined with quantitative real-time polymerase chain reaction (qPCR) to detect Akebiae Caulis in Longdan Xiegan Wan (LDXGW) for the first time. Compared with chromatographic studies, NGS enables better evaluation of the ingredient components of traditional Chinese medicine (TCM) preparations. The feasibility of qPCR using species-specific primers to determine the authenticity of species has been validated. In this study, the constituents of Akebiae Caulis in LDXGW from three different manufacturers were scanned by NGS. The independently developed qPCR detection primers of Akebiae Caulis, Aristolochiae Manshuriensis Caulis, and Clematidis Armandii Caulis were specifically used to analyze the LDXGW mentioned above.ResultsThe results showed that qPCR detected Clematidis Armandii Caulis in all commercial samples. Meanwhile, NGS detected the counterfeit species Clematis peterae (Tie-Xian Lian) in all samples. We found that qPCR shows a difference in detecting Akebiae Caulis, but it was not able to identify the unknown additives and adulterants for the primer pairs of Clematidis Armandii Caulis.ConclusionsHence, it is sensitive and rapid, qPCR is not suitable for detection alone. The NGS approaches offer important novel insights that complement the qPCR method. The combination of NGS and qPCR will be a powerful complement to traditional identification methods of TCM substances.

Genetic characterisation of a novel reptarenavirus detected in a dead pet red-tailed boa (Boa constrictor).

Boid inclusion body disease (BIBD) is a severe and transmissible disease of snakes worldwide. Reptarenaviruses have been identified as the aetiological agents of BIBD. We determined the almost complete genome sequence of an arenavirus detected in a female red-tailed boa that had succumbed in a private collection in Hungary. We used a combination of next generation sequencing and Sanger sequencing methods. Based on the analysis of the obtained sequence data, the virus, tentatively named Coldvalley virus, seemed to belong to the Reptarenavirus genus of the Arenaviridae family. This classification was confirmed by the genome structure (bisegmented single-stranded RNA) characteristic of the genera Mammarenavirus and Reptarenavirus. The pairwise comparison of the nucleotide and amino acid sequences, as well as the topology of the maximum likelihood phylogenetic trees, suggested that the newly-characterised Coldvalley virus can be classified into the species Rotterdam reptarenavirus.

STEM-07. PRC2 MAINTAINS A CANCER STEM CELL PHENOTYPE IN GLIOBLASTOMA MULTIFORME VIA CHROMATIN MODIFICATION OF H3K27me3

Abstract Multi-potent stem-like cells (i.e. cancer stem cells, CSCs) are critical determinants of tumor propagation, therapeutic resistance, and recurrence in glioblastoma (GBM). Modifications in chromatin architecture play a fundamental role in the tumor cell phenotype of GBM. The polycomb repressor complex 2 (PRC2) is a key histone modifier that supports multi-potency and oncogenesis via H3K27 trimethylation (H3K27me3). Understanding how these epigenetic modifications cooperatively drive cancer cell stemness should unveil new targets for therapeutic development in GBM. Using a combination of next-generation sequencing, bioinformatics, and molecular approaches we identified EZH2, the catalytic domain of the PRC2 complex, as a critical mediator of reprograming events in GBM cells. We found that EZH2 is highly induced in response to transgenic Oct4/Sox2 with global increases in H3K27me3. Pharmacological inhibition of EZH2 diminishes self-renewal capacity of GBM neurospheres concurrent with a reduction in gene expression levels of markers and drivers of stemness. Furthermore, we identified and validated a set of 6 putative tumor suppressor genes repressed by Oct4 and Sox2 in a PRC2-dependent manner. We identified miR-217 as an EZH2 regulator in GBM cells and miR-217 reconstitution using advanced nanoparticle formulations re-activates the PRC2-repressed tumor suppressors, inhibited tumor growth and enhanced the effects of ionizing radiation in an orthotopic model of GBM. Taken together, these data show that PRC2-mediated chromatin changes in H3K27me3 help regulate the stem-cell phenotype induced by Oct4 and Sox2 in GBM cells and predict that targeting EZH2 could have therapeutic benefit in GBM.

TERT Copy Number Alterations, Promoter Mutations and Rearrangements in Adrenocortical Carcinomas

Molecular characterization of adrenocortical carcinomas (ACC) by The Cancer Genome Atlas (TCGA) has highlighted a high prevalence of TERT alterations, which are associated with disease progression. Herein, 78 ACC were profiled using a combination of next generation sequencing (n = 76) and FISH (n = 9) to assess for TERT alterations. This data was combined with TCGA dataset (n = 91). A subset of borderline adrenocortical tumors (n = 5) and adrenocortical adenomas (n = 7) were also evaluated. The most common alteration involving the TERT gene involved gains/amplifications, seen in 22.2% (37/167) of cases. In contrast, “hotspot” promoter mutations (C > T promoter mutation at position -124, 7/167 cases, 4.2%) and promoter rearrangements (2/165, 1.2%) were rare. Recurrent co-alterations included 22q copy number losses seen in 24% (9/38) of cases. Although no significant differences were identified in cases with and without TERT alterations pertaining to age at presentation, tumor size, weight, laterality, mitotic index and Ki67 labeling, cases with TERT alterations showed worse outcomes. Metastatic behavior was seen in 70% (28/40) of cases with TERT alterations compared to 51.2% (65/127, p = 0.04) of cases that lacked these alterations. Two (of 5) borderline tumors showed amplifications and no TERT alterations were identified in 7 adenomas. In the borderline group, 0 (of 4) patients with available follow up had adverse outcomes. We found that TERT alterations in ACC predominantly involve gene amplifications, with a smaller subset harboring “hotspot” promoter mutations and rearrangements, and 70% of TERT-altered tumors are associated with metastases. Prospective studies are needed to validate the prognostic impact of these findings.

FOXL2 mutation is prevalent in metastatic adult granulosa cell tumors and is associated with improved survival

<h3>Objectives:</h3> Adult granulosa cell tumors (AGCT) of the ovary represent less than 5% of all ovarian tumors. Despite having a favorable prognosis, nearly one-third of women with this diagnosis will recur, and therapeutic options are limited. We aim to describe a molecular profile of AGCT to improve prognostication and elucidate actionable molecular targets. <h3>Methods:</h3> AGCT samples were analyzed using a combination of next-generation sequencing (NGS), immunohistochemistry (IHC) and whole transcriptome sequencing (NovaSeq) (Caris Life Sciences, Phoenix, AZ). PD-L1 expression was tested by SP-142 (Ventana; positive cut-off ≥1%). Microsatellite instability (MSI) was tested by fragment analysis, IHC and NGS. Tumor mutational burden (TMB) was measured by counting all somatic mutations per tumor (TMB-high cut-off ≥10 mutations per MB). Immune cell fraction was calculated by QuantiSeq (Finotello 2019, Genome Medicine). Gene Set Enrichment Analysis (GSEA) was used to determine enrichment of cancer hallmark pathways (Broad Institute). Survival analyses were performed using the Kaplan-Meier estimate. <h3>Results:</h3> Of the 319 patients, the median age was 55, and most patients had metastatic disease (82.4 %). A majority of the tumors carried FOXL2 mutations (83.6%), of which 90% demonstrated a <i>C134W</i> missense mutation. Patients with FOXL2 mutations were significantly older (56 versus 47 years, p=0.003) and more likely to have metastatic disease than patients with wild-type FOXL2 (p=0.045). Other frequent mutations included KMT2D (14%) and PIK3CA (8%) mutations. There were no pathogenic <i>BRCA1</i> or <i>BRCA2</i> mutations identified in this cohort; however, a <i>BRCA2</i> mutation variant of unknown significance was noted in three tumors. Pathway analyses demonstrated that alterations in chromatin remodeling were significantly more common in tumors with FOXL2 mutations compared to wild-type status (16.1% versus 9.7%, p<0.05), as were alterations in the PI3K pathway (13.9% versus 0%, p<0.05). There was a low frequency of PD-L1 high expression (2.7%), and there were no tumors with high TMB or MSI. Patients with tumors with FOXL2 mutations had improved overall survival compared to patients with wild-type FOXL2 tumors (HR 2.07, CI 1.01-4.25, p=0.043). KMT2D and PI3KCA mutations were not associated with differences in overall survival when compared to wild-type tumors. Copy number alterations in the genes GPHN, HOOK3, SPEN, CAMTA1 and BCL2L11 were associated with survival differences in patients treated with bevacizumab. <h3>Conclusions:</h3> A majority of AGCT carry FOXL2 mutations. The next most common mutations were KMT2D, PIK3CA, and alterations in chromatin remodeling. In this cohort of patients, the FOXL2 mutation was associated with improved overall survival. Copy number alterations in specific genes were associated with differences in survival in patients treated with bevacizumab. These results support molecular profiling of AGCT to improve prognostication for these patients. These findings provide groundwork to direct further investigation into novel treatment strategies and targeted agents in AGCT.