High-throughput sequencing-based analysis of the composition and diversity of the endophyte community in roots of Stellera chamaejasme.

Stellera chamaejasme (S. chamaejasme) is an important medicinal plant with heat-clearing, detoxifying, swelling and anti-inflammatory effects. At the same time, it is also one of the iconic plants of natural grassland degradation in northwest China, playing a key role in the invasion process. Plant endophytes live in healthy plant tissues and can synthesize substances needed for plant growth, induce disease resistance in host plants, and enhance plant resistance to environmental stress. Therefore, studying the root endophytes of S. chamaejasme is of great significance for mining beneficial microbial resources and biological prevention and control of S. chamaejasme. This study used Illumina MiSeq high-throughput sequencing technology to analyze the composition and diversity of endophytes in the roots of S. chamaejasme in different alpine grasslands (BGC, NMC and XGYZ) in Tibet. Research results show that the main phylum of endophytic fungi in the roots of S. chamaejasme in different regions is Ascomycota, and the main phyla of endophytic bacteria are Actinobacteria, Proteobacteria and Firmicutes (Bacteroidota). Overall, the endophyte diversity of the NMC samples was significantly higher than that of the other two sample sites. Principal coordinate analysis (PCoA) and permutational multivariate analysis of variance (PERMANOVA) results showed significant differences in the composition of endophytic bacterial and fungal communities among BGC, NMC and XGYZ samples. Co-occurrence network analysis of endophytes showed that there were positive correlations between fungi and some negative correlations between bacteria, and the co-occurrence network of bacteria was more complex than that of fungi. In short, this study provides a vital reference for further exploring and utilizing the endophyte resources of S. chamaejasme and an in-depth understanding of the ecological functions of S. chamaejasme endophytes.

Antimicrobial resistance prediction by clinical metagenomics in pediatric severe pneumonia patients.

BACKGROUND: Antimicrobial resistance (AMR) is a major threat to children's health, particularly in respiratory infections. Accurate identification of pathogens and AMR is crucial for targeted antibiotic treatment. Metagenomic next-generation sequencing (mNGS) shows promise in directly detecting microorganisms and resistance genes in clinical samples. However, the accuracy of AMR prediction through mNGS testing needs further investigation for practical clinical decision-making. METHODS: We aimed to evaluate the performance of mNGS in predicting AMR for severe pneumonia in pediatric patients. We conducted a retrospective analysis at a tertiary hospital from May 2022 to May 2023. Simultaneous mNGS and culture were performed on bronchoalveolar lavage fluid samples obtained from pediatric patients with severe pneumonia. By comparing the results of mNGS detection of microorganisms and antibiotic resistance genes with those of culture, sensitivity, specificity, positive predictive value, and negative predictive value were calculated. RESULTS: mNGS detected bacterial in 71.7% cases (86/120), significantly higher than culture (58/120, 48.3%). Compared to culture, mNGS demonstrated a sensitivity of 96.6% and a specificity of 51.6% in detecting pathogenic microorganisms. Phenotypic susceptibility testing (PST) of 19 antibiotics revealed significant variations in antibiotics resistance rates among different bacteria. Sensitivity prediction of mNGS for carbapenem resistance was higher than penicillins and cephalosporin (67.74% vs. 28.57%, 46.15%), while specificity showed no significant difference (85.71%, 75.00%, 75.00%). mNGS also showed a high sensitivity of 94.74% in predicting carbapenem resistance in Acinetobacter baumannii. CONCLUSIONS: mNGS exhibits variable predictive performance among different pathogens and antibiotics, indicating its potential as a supplementary tool to conventional PST. However, mNGS currently cannot replace conventional PST.

Characterisation of the novel HLA-B*58:02:04 allele by next-generation sequencing.

HLA-B*58:02:04 differs from HLA-B*58:02:01 by one synonymous nucleotide in codon 215 in exon 4.

KSNP: a fast de Bruijn graph-based haplotyping tool approaching data-in time cost.

Long reads that cover more variants per read raise opportunities for accurate haplotype construction, whereas the genotype errors of single nucleotide polymorphisms pose great computational challenges for haplotyping tools. Here we introduce KSNP, an efficient haplotype construction tool based on the de Bruijn graph (DBG). KSNP leverages the ability of DBG in handling high-throughput erroneous reads to tackle the challenges. Compared to other notable tools in this field, KSNP achieves at least 5-fold speedup while producing comparable haplotype results. The time required for assembling human haplotypes is reduced to nearly the data-in time.

Next-generation sequencing of host genetics risk factors associated with COVID-19 severity and long-COVID in Colombian population.

Coronavirus disease 2019 (COVID-19) was considered a major public health burden worldwide. Multiple studies have shown that susceptibility to severe infections and the development of long-term symptoms is significantly influenced by viral and host factors. These findings have highlighted the potential of host genetic markers to identify high-risk individuals and develop target interventions to reduce morbimortality. Despite its importance, genetic host factors remain largely understudied in Latin-American populations. Using a case-control design and a custom next-generation sequencing (NGS) panel encompassing 81 genetic variants and 74 genes previously associated with COVID-19 severity and long-COVID, we analyzed 56 individuals with asymptomatic or mild COVID-19 and 56 severe and critical cases. In agreement with previous studies, our results support the association between several clinical variables, including male sex, obesity and common symptoms like cough and dyspnea, and severe COVID-19. Remarkably, thirteen genetic variants showed an association with COVID-19 severity. Among these variants, rs11385942 (p < 0.01; OR = 10.88; 95% CI = 1.36-86.51) located in the LZTFL1 gene, and rs35775079 (p = 0.02; OR = 8.53; 95% CI = 1.05-69.45) located in CCR3 showed the strongest associations. Various respiratory and systemic symptoms, along with the rs8178521 variant (p < 0.01; OR = 2.51; 95% CI = 1.27-4.94) in the IL10RB gene, were significantly associated with the presence of long-COVID. The results of the predictive model comparison showed that the mixed model, which incorporates genetic and non-genetic variables, outperforms clinical and genetic models. To our knowledge, this is the first study in Colombia and Latin-America proposing a predictive model for COVID-19 severity and long-COVID based on genomic analysis. Our study highlights the usefulness of genomic approaches to studying host genetic risk factors in specific populations. The methodology used allowed us to validate several genetic variants previously associated with COVID-19 severity and long-COVID. Finally, the integrated model illustrates the importance of considering genetic factors in precision medicine of infectious diseases.

Characterization of the novel HLA-B*40:538 allele by next-generation sequencing.

The HLA-B*40:538 allele differs from HLA-B*40:01:02:01 at position 905 C→T in exon 5.

Development, testing and validation of a targeted NGS-panel for the detection of actionable mutations in lung cancer (NSCLC) using anchored multiplex PCR technology in a multicentric setting.

Lung cancer is a paradigm for a genetically driven tumor. A variety of drugs were developed targeting specific biomarkers requiring testing for tumor genetic alterations in relevant biomarkers. Different next-generation sequencing technologies are available for library generation: 1) anchored multiplex-, 2) amplicon based- and 3) hybrid capture-based-PCR. Anchored multiplex PCR-based sequencing was investigated for routine molecular testing within the national Network Genomic Medicine Lung Cancer (nNGM). Four centers applied the anchored multiplex ArcherDX-Variantplex nNGMv2 panel to re-analyze samples pre-tested during routine diagnostics. Data analyses were performed by each center and compiled centrally according to study design. Pre-defined standards were utilized, and panel sensitivity was determined by dilution experiments. nNGMv2 panel sequencing was successful in 98.9% of the samples (N = 90). With default filter settings, all but two potential MET exon 14 skipping variants were identified at similar allele frequencies. Both MET variants were found with an adapted calling filter. Three additional variants (KEAP1, STK11, TP53) were called that were not identified in pre-testing analyses. Only total DNA amount but not a qPCR-based DNA quality score correlated with average coverage. Analysis was successful with a DNA input as low as 6.25 ng. Anchored multiplex PCR-based sequencing (nNGMv2) and a sophisticated user-friendly Archer-Analysis pipeline is a robust and specific technology to detect tumor genetic mutations for precision medicine of lung cancer patients.

Clinical performance of metagenomic next-generation sequencing for diagnosis of pulmonary Aspergillus infection and colonization.

Background: Investigations assessing the value of metagenomic next-generation sequencing (mNGS) for distinguish Aspergillus infection from colonization are currently insufficient. Methods: The performance of mNGS in distinguishing Aspergillus infection from colonization, along with the differences in patients' characteristics, antibiotic adjustment, and lung microbiota, were analyzed. Results: The abundance of Aspergillus significantly differed between patients with Aspergillus infection (n=36) and colonization (n=32) (P < 0.0001). Receiver operating characteristic (ROC) curve result for bronchoalveolar lavage fluid (BALF) mNGS indicated an area under the curve of 0.894 (95%CI: 0.811-0.976), with an optimal threshold value of 23 for discriminating between Aspergillus infection and colonization. The infection group exhibited a higher proportion of antibiotic adjustments in comparison to the colonization group (50% vs. 12.5%, P = 0.001), with antibiotic escalation being more dominant. Age, length of hospital stay, hemoglobin, cough and chest distress were significantly positively correlated with Aspergillus infection. The abundance of A. fumigatus and Epstein-Barr virus (EBV) significantly increased in the infection group, whereas the colonization group exhibited higher abundance of A. niger. Conclusion: BALF mNGS is a valuable tool for differentiating between colonization and infection of Aspergillus. Variations in patients' age, length of hospital stay, hemoglobin, cough and chest distress are observable between patients with Aspergillus infection and colonization.

Proceedings of the 2023 Viral Clearance Symposium, Session 8: Cell Banks, Advanced Technologies (ATMPs, NGS).

The Cell Banks, Advanced Technologies (ATMPs, NGS) session at the 2023 Viral Clearance Symposium (VCS) focused on the assurance of high virus safety profiles of advanced technology medicinal products (ATMPs) by implementation of advanced virus detection methods using rapid and sensitive technologies, such as next-generation sequencing (NGS). All presentations in this session made the need to replace in vivo testing for viruses by new technologies that have been demonstrated to be incomparably broad in their detection capabilities and can even detect unknown viruses. An evaluation of historical data collected by the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) from their members' in vivo and in vitro adventitious virus test experience as well as on using NGS was presented. The data convincingly supported the necessity to replace in vivo testing with faster, broader, more sensitive, more accurate, and more specific virus detection methods. Additionally, a collaborative study-initiated by the CAACB-with the goal to revisit traditional adventitious agent testing by using targeted NGS to replace in vivo and in vitro tests for well-known and broadly used Chinese hamster ovary (CHO) cells was presented, including the planned risk-assessment approach using prior knowledge and historical data. Overall, this session demonstrated that the use of new virus detection methods, such as NGS, represents a great opportunity to provide sufficient viral safety margins, specifically, for ATMPs, where downstream virus clearance is not possible. This path forward is also supported by the final ICH Q5A(R2) guideline.

Proceedings of the 2023 Viral Clearance Symposium: 2023 VCS Summary, Pending Questions, and Next Steps.

The 2023 Viral Clearance Symposium (VCS) was hosted by Takeda on 24 and 25 May 2023 in Vienna, Austria. The present conference extended the structure of the previous biennial symposia held between 2009 and 2019. As recapitulated in the introductory session, the genesis of the VCS, as described in the Proceedings of the 2009 VCS was "the worldwide regulatory and industry recognition that challenges, gaps, and opportunities exist, that it formally addressed could benefit the field as whole." This report provides a synopsis of the progress achieved at the conference resulting from detailed technical discussions and the pending questions that still require attention to address. The 2023 VCS was composed of nine individual sessions of short presentations followed by in-depth panel discussions from the presenters. Sessions included Regulatory Updates (with a focus on ICH Q5A(R2) efforts), including a summary of lessons learned from the 2019 VCS, and progress on these key areas mapped into 2023 VCS topics: Viral Clearance Strategy and Case Studies, New Modalities in Chromatography and Adsorptive Filters, Continuous Processing, Viral Clearance Strategy and Process Understanding, Virus Inactivation, Upstream and Downstream Virus Retentive Filtration and Cell Banks, and Advanced Technologies (advanced therapy medicinal products, next-generation sequencing).

Mutation landscape in Chinese nodal diffuse large B-cell lymphoma by targeted next generation sequencing and their relationship with clinicopathological characteristics.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL), an aggressive and heterogenic malignant entity, is still a challenging clinical problem, since around one-third of patients are not cured with primary treatment. Next-generation sequencing (NGS) technologies have revealed common genetic mutations in DLBCL. We devised an NGS multi-gene panel to discover genetic features of Chinese nodal DLBCL patients and provide reference information for panel-based NGS detection in clinical laboratories. METHODS: A panel of 116 DLBCL genes was designed based on the literature and related databases. We analyzed 96 Chinese nodal DLBCL biopsy specimens through targeted sequencing. RESULTS: The most frequently mutated genes were KMT2D (30%), PIM1 (26%), SOCS1 (24%), MYD88 (21%), BTG1 (20%), HIST1H1E (18%), CD79B (18%), SPEN (17%), and KMT2C (16%). SPEN (17%) and DDX3X (6%) mutations were highly prevalent in our study than in Western studies. Thirty-three patients (34%) were assigned as genetic classification by the LymphGen algorithm, including 12 cases MCD, five BN2, seven EZB, seven ST2, and two EZB/ST2 complex. MYD88 L265P mutation, TP53 and BCL2 pathogenic mutations were unfavorable prognostic biomarkers in DLBCL. CONCLUSIONS: This study presents the mutation landscape in Chinese nodal DLBCL, highlights the genetic heterogeneity of DLBCL and shows the role of panel-based NGS to prediction of prognosis and potential molecular targeted therapy in DLBCL. More precise genetic classification needs further investigations.

Direct Determination of the Structure of Single Biopolymer Molecules Using Nanopore Sequencing.

This review highlights operational principles, features, and modern aspects of the development of third-generation sequencing technology of biopolymers focusing on the nucleic acids analysis, namely the nanopore sequencing system. Basics of the method and technical solutions used for its realization are considered, from the first works showing the possibility of creation of these systems to the easy-to-handle procedure developed by Oxford Nanopore Technologies company. Moreover, this review focuses on applications, which were developed and realized using equipment developed by the Oxford Nanopore Technologies, including assembly of whole genomes, methagenomics, direct analysis of the presence of modified bases.

Detection of SARS-COV-2 variants and their proportions in wastewater samples using next-generation sequencing in Finland.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants may have different characteristics, e.g., in transmission, mortality, and the effectiveness of vaccines, indicating the importance of variant detection at the population level. Wastewater-based surveillance of SARS-CoV-2 RNA fragments has been shown to be an effective way to monitor the COVID-19 pandemic at the population level. Wastewater is a complex sample matrix affected by environmental factors and PCR inhibitors, causing insufficient coverage in sequencing, for example. Subsequently, results where part of the genome does not have sufficient coverage are not uncommon. To identify variants and their proportions in wastewater over time, we utilized next-generation sequencing with the ARTIC Network's primer set and bioinformatics pipeline to evaluate the presence of variants in partial genome data. Based on the wastewater data from November 2021 to February 2022, the Delta variant was dominant until mid-December in Helsinki, Finland's capital, and thereafter in late December 2022 Omicron became the most common variant. At the same time, the Omicron variant of SARS-CoV-2 outcompeted the previous Delta variant in Finland in new COVID-19 cases. The SARS-CoV-2 variant findings from wastewater are in agreement with the variant information obtained from the patient samples when visually comparing trends in the sewerage network area. This indicates that the sequencing of wastewater is an effective way to monitor temporal and spatial trends of SARS-CoV-2 variants at the population level.

Quality Control of an Isogenic H/N/KRAS-Less Mouse Embryonic Fibroblast Cell Line Panel.

Isogenic H/N/KRAS-less mouse embryonic fibroblast (MEF) cell lines have been developed to assist in cell-based assays of RAS inhibitors. The quality control assessment of a panel of these isogenic MEFs is described here, with a focus on ensuring the proper insertion of the desired mutant RAS transgene, a determination of gene copy number, and an investigation of potential off-target mutations which could lead to phenotypes which are undesired in downstream experiments. Using this suite of quality control tools, a MEF cell line can be readily validated, and researchers can be assured of the rationale for an observed phenotype.

Case report: Primary vulvar adenocarcinoma of mammary gland type-its genetic characteristics by focused next-generation sequencing.

Mammary-like vulvar adenocarcinoma (MLVA) is an exceedingly rare subtype of vulvar adenocarcinoma that shares features with mammary gland tissue. Due to its rarity and lack of consensus, MLVA presents diagnostic challenges to pathologists. We present the case of a 59-year-old female with an ulcerated mass on the right side of the external genitalia, diagnosed as MLVA. Comprehensive immunohistochemistry (IHC) and gene sequencing studies were performed to characterize the tumor. IHC analysis revealed triple expression of hormonal receptors (estrogen receptor, progesterone receptor, and HER2), supporting the mammary gland origin of the tumor. Gene sequencing identified unique genetic mutations associated with the expression of hormonal markers. One fusion gene (ERBB2-NAGLU) has not been reported in any tumors, and other mutations with unique mutation types have not been previously reported in MLVA. Our findings shed light on the molecular characteristics of MLV and may help improve the diagnosis and treatment of this rare type of vulvar adenocarcinoma.

Detection of the HLA-C*07:04:29 allele in a Chinese individual.

HLA-C*07:04:29 differs from HLA-C*07:04:01:01 by a single substitution in exon 4.

Identification of the novel HLA-C allele, HLA-C*07:02:150.

A novel HLA-C*07 allele, now officially designated HLA-C*07:02:150, was identified by next-generation sequencing.

Combination of metagenomic next-generation sequencing and conventional tests unraveled pathogen profiles in infected patients undergoing allogeneic hematopoietic stem cell transplantation in Jilin Province of China.

Background: Infection is the main cause of death for patients after allogeneic hematopoietic stem cell transplantation (HSCT). However, pathogen profiles still have not been reported in detail due to their heterogeneity caused by geographic region. Objective: To evaluate the performance of metagenomic next-generation sequencing (mNGS) and summarize regional pathogen profiles of infected patients after HSCT. Methods: From February 2021 to August 2022, 64 patients, admitted to the Department of Hematology of The First Hospital of Jilin University for HSCT and diagnosed as suspected infections, were retrospectively enrolled. Results: A total of 38 patients were diagnosed as having infections, including bloodstream (n =17), pulmonary (n =16), central nervous system (CNS) (n =4), and chest (n =1) infections. Human betaherpesvirus 5 (CMV) was the most common pathogen in both bloodstream (n =10) and pulmonary (n =8) infections, while CNS (n =2) and chest (n =1) infections were mainly caused by Human gammaherpesvirus 4 (EBV). For bloodstream infection, Mycobacterium tuberculosis complex (n =3), Staphylococcus epidermidis (n =1), and Candida tropicalis (n =1) were also diagnosed as causative pathogens. Furthermore, mNGS combined with conventional tests can identify more causative pathogens with high sensitivity of 82.9% (95% CI 70.4-95.3%), and the total coincidence rate can reach up to 76.7% (95% CI 64.1-89.4%). Conclusions: Our findings emphasized the importance of mNGS in diagnosing, managing, and ruling out infections, and an era of more rapid, independent, and impartial diagnosis of infections after HSCT can be expected.

The novel HLA-C*03:94:02 allele identified by next-generation sequencing in a Chinese individual.

HLA-C*03:94:02 differs from HLA-C*03:94:01 by a single nucleotide substitution in exon 2 (codon 17 GGA->GGG).