Sequencing Strategy Research Articles

Metabolomic reprogramming of subcutaneous adipose tissue aggravates atherosclerosis via impaired adipocyte-macrophage crosstalk

Abstract Object: The bidirectional relationship between cardiometabolic syndrome and dysfunctional adipose tissue has received increasing attention. However, the metabolic alteration of subcutaneous adipose tissue during atherosclerosis has been overlooked. Methods In this study, we aimed to investigate the relationship between subcutaneous adipose tissue and atherosclerosis based on a combination strategy of single-cell sequencing and metabolomics in atherosclerotic mice and humans. Result Through our analysis of the clinical cohort with atherosclerosis, we discovered a correlation between the volume of subcutaneous adipose tissue and the characteristics of coronary plaques. Furthermore, by employing the snRNA-seq approach, we identified the metabolic landscape and cellular crosstalk within the subcutaneous adipose tissue of atherosclerotic mice. Lastly, through our analysis of the metabolic features in human adipose tissue, we successfully validated the Insulin-like Growth Factor 1 pathway as a crucial modulator in the adipocyte-macrophage crosstalk during the development of atherosclerosis. Conclusion The present study has provided further evidence that dysregulated metabolism of subcutaneous adipose tissue is involved in the development and progression of atherosclerosis, not only in humans but also in animal models.

Pharmacotherapy sequencing strategies for patients with heart failure with reduced ejection fraction: a cost–utility analysis

Pharmacotherapy sequencing strategies for patients with heart failure with reduced ejection fraction: a cost–utility analysis

Unravelling the architecture of major histocompatibility complex class II haplotypes in rhesus macaques.

The regions in the genome that encode components of the immune system are often featured by polymorphism, copy number variation, and segmental duplications. There is a need to thoroughly characterize these complex regions to gain insight into the impact of genomic diversity on health and disease. Here we resolve the organization of complete major histocompatibility complex (MHC) class II regions in rhesus macaques by using a long-read sequencing strategy (Oxford Nanopore Technologies) in concert with adaptive sampling. In particular, the expansion and contraction of the primate DRB-region appear to be a dynamic process that involves the rearrangement of different cassettes of paralogous genes. These chromosomal recombination events are propagated by a conserved pseudogene, DRB6, which features the integration of two retroviral elements. In contrast, the DRA locus appears to be protected from rearrangements, which may be owing to the presence of an adjacently located truncated gene segment, DRB9 With our sequencing strategy, the annotation, evolutionary conservation, and potential function of pseudogenes can be reassessed, an aspect that was neglected by most genome studies in primates. Furthermore, our approach facilitates the characterization and refinement of an animal model essential to study human biology and disease.

Bispecific antibodies as bridging to BCMA CAR-T cell therapy for relapsed/refractory multiple myeloma.

Establishing a strategy for sequencing of T cell redirecting therapies for relapsed/refractory multiple myeloma (RRMM) is a pressing clinical need. We longitudinally tracked the clinical and immunological impact of bispecific T cell engaging antibodies (BsAb) as bridging therapy (BT) to subsequent BCMA-directed CAR-T cell therapies in 52 RRMM patients. BsAbs were a potent and safe option for BT, achieving the highest overall response rate (100%) to BT compared to chemotherapy, anti-CD38 or anti-SLAMF7 antibody based regimens (46%). We observed early CD4+CAR+ and delayed CD8+CAR+ T cell expansion in patients receiving BsAb as BT. In vitro cytotoxicity of CAR-T cells was comparable amongst BT options. Single-cell analyses revealed increased clonality in the CD4+ and CD8+ T cell compartments in patients with previous exposure to BsAbs at leukapheresis and on day 30 after CAR-T infusion. This study demonstrates the feasibility and efficacy of BT with BsAbs for CAR-T cell therapy in RRMM.

The Journey of Antibody-Drug Conjugates: Lessons Learned from 40 Years of Development.

Antibody-drug conjugates (ADC) represent one of the most rapidly expanding treatment modalities in oncology, with 11 ADCs approved by the FDA and more than 210 currently being tested in clinical trials. Spanning over 40 years, ADC clinical development has enhanced our understanding of the multifaceted mechanisms of action for this class of therapeutics. In this article, we discuss key insights into the toxicity, efficacy, stability, distribution, and fate of ADCs. Furthermore, we highlight ongoing challenges related to their clinical optimization, the development of rational sequencing strategies, and the identification of predictive biomarkers. Significance: The development and utilization of ADCs have allowed for relevant improvements in the prognosis of multiple cancer types. Concomitantly, the rise of ADCs in oncology has produced several challenges, including the prediction of their activity, their utilization in sequence, and minimization of their side effects, that still too often resemble those of the cytotoxic molecule that they carry. In this review, we retrace 40 years of development in the field of ADCs and delve deep into the mechanisms of action of these complex therapeutics and reasons behind the many achievements and failures observed in the field to date.

Breast Cancer Adjuvant Radiotherapy and Chemotherapy Sequencing: Sequential, Concomitant, or What Else? A Comprehensive Review of the Adjuvant Combinations Journey.

Background: To date, in breast cancer (BC) treatment, adjuvant chemotherapy (A-CT) has preceded adjuvant radiotherapy (A-RT). In the last twenty years, the adjuvant treatment of BC has quickly evolved due to better knowledge of its molecular biology, genetic profile, and α/β ratio of 3/4 Gy for tumor and normal tissue radiosensitivity. Thus, new schedules with hypofractionated radiotherapy have been tested, and a third generation of A-CT has been introduced, raising the question of whether it is time to rethink the sequencing between these two approaches. Methods: In the last 20 years, many attempts have been made worldwide to optimize the best sequencing strategy between these two approaches in terms of sequential CT-RT and RT-CT and concomitant and sandwich modalities using drugs and schedules. This paper presents a comprehensive review of the state of the art, analyzing all the available studies to assess the sequencing between A-CT and A-RT with different generations of chemotherapy schedules. Results: More than 8000 patients from 30 studies treated with adjuvant chemotherapy and whole breast radiotherapy who were enrolled in randomized, retrospective, and prospective studies were analyzed. Sequential, concomitant, and sandwich modalities of chemotherapy with conventional or hypofractionated RT schedules from the most important studies were included. The most used sequence was adjuvant chemotherapy followed by conventional or hypofractionated radiotherapy. In the concomitant approach, i.v. CMF has been the most important adopted schedule, while the concomitant use of anthracyclines and taxanes with conventional or hypofractionated radiotherapy has been found to be more toxic. One study analyzed the benefit in terms of reducing adjuvant treatment time with upfront hypofractionated radiotherapy and third-generation chemotherapy. Conclusions: At present, the best sequencing strategy has not yet been defined. This comprehensive review is a journey among the most important randomized, retrospective, and prospective studies that highlights the past, current, and novel time sequencing proposals between A-CT and A-RT to assess the state of the art and provide useful information for future adjuvant approaches in breast cancer treatment.

Unraveling the developmental heterogeneity within the human retina to reconstruct the continuity of retinal ganglion cell maturation and stage-specific intrinsic and extrinsic factors.

Tissue development is a complex spatiotemporal process with multiple interdependent components. Anatomical, histological, sequencing, and evolutional strategies can be used to profile and explain tissue development from different perspectives. The introduction of scRNAseq methods and the computational tools allows to deconvolute developmental heterogeneity and draw a decomposed uniform map. In this manuscript, we decomposed the development of a human retina with a focus on the retinal ganglion cells (RGC). To increase the temporal resolution of retinal cell classes maturation state we assumed the working hypothesis that that maturation of retinal ganglion cells is a continuous, non-discrete process. We have assembled the scRNAseq atlas of human fetal retina from fetal week 8 to week 27 and applied the computational methods to unravel maturation heterogeneity into a uniform maturation track. We align RGC transcriptomes in pseudotime to map RGC developmental fate trajectories against the broader timeline of retinal development. Through this analysis, we identified the continuous maturation track of RGC and described the cell-intrinsic (DEGs, maturation gene profiles, regulons, transcriptional motifs) and -extrinsic profiles (neurotrophic receptors across maturation, cell-cell interactions) of different RGC maturation states. We described the genes involved in the retina and RGC maturation, including de novo RGC maturation drivers. We demonstrate the application of the human fetal retina atlas as a reference tool, allowing automated annotation and universal embedding of scRNAseq data. Altogether, our findings deepen the current knowledge of the retina and RGC maturation by bringing in the maturation dimension for the cell class vs. state analysis. We show how the pseudotime application contributes to developmental-oriented analyses, allowing to order the cells by their maturation state. This approach not only improves the downstream computational analysis but also provides a true maturation track transcriptomics profile.

An estimation of global genetic prevalence of PLA2G6-associated neurodegeneration

BackgroundPLA2G6-associated neurodegeneration (PLAN) comprises three diseases with overlapping features: infantile neuroaxonal dystrophy (INAD), atypical neuroaxonal dystrophy (atypical NAD), and PLA2G6-related dystonia-parkinsonism. INAD is an early onset disease characterized by progressive loss of vision, muscular control, and mental skills. The prevalence of PLA2G6-associated diseases has not been previously calculated.MethodsTo provide the most accurate prevalence estimate, we utilized two independent approaches: database-based approach which included collecting variants from ClinVar, Human Gene Mutation Database (HGMD) and high confidence predicted loss-of-function (pLoF) from gnomAD (Rare Genomes Project Genetic Prevalence Estimator; GeniE), and literature-based approach which gathered variants through Mastermind Genomic Search Engine (Genomenon, Inc). Genetic prevalence of PLAN was calculated based on allele frequencies from gnomAD, assuming Hardy–Weinberg equilibrium.ResultsIn the PLA2G6 gene, our analysis found 122 pathogenic, 82 VUS, and 15 variants with conflicting interpretations (pathogenic vs VUS) between two approaches. Allele frequency was available for 58 pathogenic, 42 VUS, and 15 conflicting variants in gnomAD database. If pathogenic and/or conflicting variants are included, the overall genetic prevalence was estimated to be between 1 in 987,267 to 1 in 1,570,079 pregnancies, with the highest genetic prevalence in African/African-American (1 in 421,960 to 1 in 365,197) and East-Asian (1 in 683,978 to 1 in 190,771) populations.ConclusionOur estimates highlight the significant underdiagnosis of PLA2G6-associated neurodegeneration and underscores the need for increased awareness and diagnostic efforts. Furthermore, our study revealed a higher carrier frequency of PLA2G6 variants in African and Asian populations, stressing the importance of expanded genetic sequencing in non-European populations to ensure accurate and comprehensive diagnosis. Future research should focus on confirming our findings and implementing expanded sequencing strategies to facilitate maximal and accurate diagnosis, particularly in non-European populations.

7644 Novel Candidate Genes for SRY-Negative 46,XX Differences of Sex Development with Testicular Differentiation

Abstract Disclosure: M.M. Ferrari: None. M.Y. Nishi: None. A.A. Jorge: None. A.F. Benedetti,: None. R.L. Batista: None. E.S. Silva: None. A. Martinez: None. V. Mericq: None. F.M. Carvalho: None. B.B. Mendonca: None. S. Domenice: None. Introduction: Gonadal development in mammals is a dynamic and complex process, which requires the interaction of multiple factors in a meticulous feedback control and self-regulation system. Defects in genes involved in this process may cause differences of sex development (DSD). In rare cases, the gonads of 46,XX individuals differentiate completely into testes (46,XX testicular DSD, 46,XX TDSD), or with the coexistence of testicular and ovarian tissues in the same individual (46,XX ovotesticular DSD, 46,XX OTDSD). Strategies of large-scale parallel sequencing have enabled the simultaneous analysis of known genes, as well as the identification of novel candidate genes contributing to molecular diagnosis of these conditions. Objective: To establish the molecular diagnosis of a cohort of SRY-negative 46,XX T/OTDSD patients using the whole exome sequencing (WES) technique. Patients and Methods: Twenty-three families (24 patients) were evaluated, 22 patients with sporadic disease, and one family with two affected siblings. Histological diagnosis of OTDSD was confirmed in all patients, except in two with the presumptive (non-histological) diagnosis of TDSD. WES was performed using the Illumina platform. The variants were classified according to ACMG criteria. Results: Six different candidate genes associated with the 46,XX DSD phenotype were identified in the 23 families (26%) studied; all in patients with OTDSD diagnosis. Two variants were classified as likely pathogenic in FLNB and SOX10 genes, and four as VUS in IMMP2L, SOX8, PRKACG, and CLASP1 genes. The unavailability of analysis of case-parent trio samples hampered the classification of some of the novel candidate allelic variants. All these candidate genes were selected as potential causes of OTDSD, based on evidence of direct or indirect interactions of these proteins with SOX9. In the two siblings with 46,XX OTDSD, the allelic variant c.397delT (p.Ser133Leufs*7) in IMMP2L was identified in a heterozygous state and was absent in the unaffected family members (mother and third XY sibling). A potential role of Immp2l had been previously associated with the etiology of XX female-to-male sex reversal in sheep lineage. Conclusion: Although establishing the genetic etiology of gonadal development abnormalities in individuals with 46,XX TDSD/OTDSD remains a challenge, the results of this study generated new information and perspectives on the mechanisms involved in gonadal development. Presentation: 6/3/2024

Resolved genomes of wastewater ESBL-producing Escherichia coli and metagenomic analysis of source wastewater samples.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli pose a serious threat to human health because of their resistance to the most commonly prescribed antibiotics: penicillins and cephalosporins. In this study, we provide a genomic and metagenomic context for the determinant beta-lactam resistance genes of ESBL-positive E. coli isolated from various wastewater treatment utilities in Oregon, USA. Class A beta-lactamase genes on chromosomes (blaCTX-M, blaTEM) were clustered with antibiotic resistance genes associated with other classes of antibiotics (sulfonamides and aminoglycosides) along with insertional elements. ESBL genes such as blaCTX-M, blaTEM, and blaSHV were also detected on conjugable plasmids of IncF and IncI incompatibility types. One novel IncF plasmid (pSHV2A_ESBLF) was identified, which carried a multidrug resistance genotype (blaSHV-2A, aadA22, aac3, aph6, tetA, and sul1) in addition to a mer (mercury resistance) operon, colicin, and aerobactin genes. Shotgun metagenomic analysis of the ESBL-producing E. coli-originating wastewater samples showed the presence of class A beta-lactamases; however, the ESBL genes identified in the E. coli genomes were below the detection limits. Other ESBL-associated genes (i.e., blaOXA.11, blaFOX.7, and blaGES.17) were identified in the wastewater samples, and their occurrences were correlated with the core microbial genera (e.g., Paraprevotella). In the E. coli genomes and wastewater samples, tetracycline, aminoglycoside, and beta-lactam resistance determinants frequently co-occurred. The combination of whole-genome and metagenomic analysis provides a holistic description of ESBL-producing organisms and genes in wastewater systems.IMPORTANCEUsing a hybrid sequencing and assembly strategy (short- and long-read sequencing), we identified the distribution of ARGs and virulence factors harbored on plasmids and chromosomes. We further characterized plasmids' incompatibility types and the co-occurrences of ARGs and virulence factors on plasmids and chromosomes. We investigated the transferability of plasmid-mediated beta-lactams via conjugation. Finally, using shotgun metagenomic analysis of the ESBL-producing Escherichia coli-originated wastewater samples, we described the microbial community, the resistome composition, and the potential associations with plasmid-mediated beta-lactam genes and other ARGs.

Improving Health Outcomes Through Treatment Sequencing Optimization in Multiple Myeloma: A Simulation Model in Transplant-Ineligible Patients.

Patients with multiple myeloma often require multiple treatment lines. The order in which treatments are sequenced has impact on clinical outcomes. This study aimed to estimate progression-free survival (PFS) and overall survival (OS) with common treatment sequences used in Portugal and the incremental benefit of an optimal sequence in transplant-ineligible patients with multiple myeloma. A state-transition sequential model with a five-health state conceptual structure was developed to simulate and compare survival outcomes between treatment sequences up to four lines of treatments. Data sources included randomized clinical trials and indirect treatment comparisons. A panel of Portuguese hematologists listed four most common treatment sequences and optimal sequence of choice in transplant-ineligible patients. Our simulation estimated an OS between 6.1 and 7.8 years using the most common sequences, with VMP + DRd + Pd + Kd as the most effective (7.8 years). Optimal sequence of choice (DRd + PVd + Kd + Vd) achieved OS of 9.8 years and may extend OS in 2.0-3.7 years vs. most common sequences (26%-61% increase). This benefit was mostly explained by extended PFS in the first line of treatment. Model results demonstrate that choosing the most effective treatment upfront is crucial in delaying disease progression thus yielding better survival outcomes in transplant-ineligible patients. There was a clear survival benefit in using daratumumab-based regimens in first line. This modelling exercise highlights the need to raise awareness around the impact of sequencing strategies to improve patient's outcomes.

Optimal Sequence for Total Neoadjuvant Therapy in Locally Advanced Rectal Cancer: An Evidence-Based Review.

Historically, multimodal therapeutic strategies involving preoperative chemoradiotherapy (CRT), surgery, and adjuvant chemotherapy (CT) have been employed to treat locally advanced rectal cancer (LARC). Total Neoadjuvant Therapy (TNT) is showing promise in improving outcomes. Despite its benefits, the optimal sequencing within TNT-whether induction chemotherapy should precede or follow chemoradiotherapy-remains a critical question. This study endeavors to explore the effects of different TNT sequencing strategies on patient outcomes, including tumor downstaging, pathological response, organ preservation, and the balance between efficacy and tolerability. Our methodology entailed a comprehensive literature review conducted on Medline, focusing on recent research, including retrospective studies, systematic reviews, and clinical trials. The review emphasized the comparison of induction chemotherapy versus consolidation chemotherapy within TNT regimens, assessing outcomes such as pathological response, organ preservation rates, and adverse effects. To ensure the selection of appropriate and high-quality studies, specific inclusion and exclusion criteria were applied. The analysis revealed that induction chemotherapy might lead to decreased adherence to subsequent chemoradiotherapy while offering an early intervention against micrometastasis and potentially improving overall chemotherapy compliance. Conversely, consolidation chemotherapy has been associated with higher pathological complete response (pCR) rates and improved tolerability, indicating its potential for patients requiring local symptom relief or those eligible for a nonoperative management approach. Comparative studies like CAO/ARO/AIO-12 and the OPRA trials have significantly contributed to our understanding, suggesting that while both strategies have distinct advantages, the choice between induction and consolidation chemotherapy should be tailored based on individual patient profiles and tumor characteristics. This narrative review underscores the importance of a nuanced approach to TNT sequencing in locally advanced rectal cancer, highlighting the need for further research to refine treatment strategies.

Virtual Simulation-Based Optimization for Assembly Flow Shop Scheduling Using Migratory Bird Algorithm.

As industrial informatization progresses, virtual simulation technologies are increasingly demonstrating their potential in industrial applications. These systems utilize various sensors to capture real-time factory data, which are then transmitted to servers via communication interfaces to construct corresponding digital models. This integration facilitates tasks such as monitoring and prediction, enabling more accurate and convenient production scheduling and forecasting. This is particularly significant for flexible or mixed-flow production modes. Bionic optimization algorithms have demonstrated strong performance in factory scheduling and operations. Centered around these algorithms, researchers have explored various strategies to enhance efficiency and optimize processes within manufacturing environments.This study introduces an efficient migratory bird optimization algorithm designed to address production scheduling challenges in an assembly shop with mold quantity constraints. The research aims to minimize the maximum completion time in a batch flow mixed assembly flow shop scheduling problem, incorporating variable batch partitioning strategies. A tailored virtual simulation framework supports this objective. The algorithm employs a two-stage encoding mechanism for batch partitioning and sequencing, adapted to the unique constraints of each production stage. To enhance the search performance of the neighborhood structure, the study identifies and analyzes optimization strategies for batch partitioning and sequencing, and incorporates an adaptive neighborhood structure adjustment strategy. A competition mechanism is also designed to enhance the algorithm's optimization efficiency. Simulation experiments of varying scales demonstrate the effectiveness of the variable batch partitioning strategy, showing a 5-6% improvement over equal batch strategies. Results across different scales and parameters confirm the robustness of the algorithm.

Efficient Strategy to Discover DNA Aptamers Against Low Abundance Cell Surface Proteins in Scarce Samples.

Molecular recognition probes targeting cell surface proteins such as aptamers play crucial roles in precise diagnostics and therapy. However, the selection of aptamers against low-abundance proteins in situ on the cell surface, especially in scarce samples, remains an unmet challenge. In this study, we present a single-round, single-cell aptamer selection method by employing a digital DNA sequencing strategy, termed DiDS selection, to address this dilemma. This approach incorporates a molecular identification card for each DNA template, thereby mitigating biases introduced by multiple PCR amplifications and ensuring the accurate identification of aptamer candidates. Through DiDS selection, we successfully obtained a series of high-quality aptamers against cell lines, clinical specimens, and neurons. Subsequent analyses for target identification revealed that aptamers derived from DiDS selection exhibit recognition capabilities for proteins with varying abundance levels. In contrast, multiple rounds of selection resulted in the enrichment of only one aptamer targeting a high-abundance target. Moreover, the comprehensive profiling of cell surfaces at the single-cell level, utilizing an enriched aptamer pool, revealed unique molecular patterns for each cell line. This streamlined approach holds promise for the rapid generation of specific recognition molecules targeting cell surface proteins across a broad range of expression levels and expands its applications in cell profiling, specific probe identification, biomarker discovery, etc.

A correctable decoding DNA sequencing with high accuracy and high throughput.

Eliminating errors in next-generation sequencing has proven to be challenging. Here we present a novel strategy for DNA sequencing, called correctable two-color fluorogenic DNA decoding sequencing, which can significantly improve sequencing accuracy and throughput by employing a dual-nucleotide addition combined with fluorogenic sequencing-by-synthesis (SBS) chemistry. This sequencing method involves introducing a mixture of natural nucleotide X, labeled unblocked nucleotide X', 3' blocked nucleotide Y*, and labeled 3' blocked nucleotide Y* into each reaction cycle. By cyclically interrogating a template twice with different nucleotide combinations, two sets of base-encoding are sequentially obtained, enabling accurate deduction of base sequence. We demonstrate the remarkable efficacy of this approach in detecting and correcting sequencing errors, achieving a theoretical error rate of 0.0005%, which is twice as accurate as Sanger sequencing. Furthermore, we show the capability to detect known mutation sites using information from only a single sequencing run. The correctable two-color fluorogenic DNA decoding sequencing approach should enable accurate identification of extremely rare genomic variations in diverse applications in biology and medicine.

Elucidating the Genetic Regulation of Reproductive Prolificacy in Tibetan Sheep Through Ovarian Tissue Sequencing

Background: This study aims to unravel the genetic intricacies regulating the reproductive prolificacy of Tibetan sheep, an indigenous breed prominently found in the Qinghai-Tibet Plateau. Methods: We employed meticulous sequencing and analytical strategies to discern the differential expressions of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in the ovarian tissues of ewes with varying lambing numbers. Result: In our study, we identified 817 genes with differential expression in twin-bearing ewes compared to those bearing single lambs. This group comprised 495 upregulated and 322 downregulated genes, featuring key candidates like COL1A1, COL1A2, BMPRIB, GDF9, BMP15 and TGFB2. Furthermore, we discovered a notable difference in the expression of long non-coding RNAs (lncRNAs) in ewes birthing twins as opposed to singles, with 210 differentially expressed lncRNAs identified, including 91 upregulated and 119 downregulated. Key genes and lncRNAs associated with reproductive efficiency were identified, contributing valuable insights into the underlying molecular mechanisms and offering a robust foundation for enhancing breeding strategies and overall livestock productivity.

Evaluation of a Nanopore Sequencing Strategy on Bacterial Communities From Marine Sediments

ABSTRACTFollowing the development of high‐throughput DNA sequencers, environmental prokaryotic communities were usually described by metabarcoding on short markers of the 16S domain. Among third‐generation sequencers, that offered the possibility to sequence the full 16S domain, the portable MinION from Oxford Nanopore was undervalued for metabarcoding because of its relatively higher error rate per read. Here we illustrate the limits and benefits of Nanopore sequencing devices by comparing the prokaryotic community structure in a mock community and 52 sediment samples from mangrove sites, inferred from full‐length 16S long‐reads (16S‐FL, ca. 1.5 kbp) on a MinION device, with those inferred from partial 16S short‐reads (16S‐V4V5, ca. 0.4 kbp, 16S‐V4V5) on Illumina MiSeq. 16S‐V4V5 and 16S‐FL retrieved all the bacterial species from the mock, but Nanopore long‐reads overestimated their diversity (56 species vs. 15). Whether these supplementary OTUs were artefactual or not, they only accounted for ca. 10% of the reads. From the sediment samples, with a coverage‐based rarefaction of reads and after singletons filtering, Mantel and Procrustean tests of co‐inertia showed that bacterial community structures inferred from 16S‐V4V5 and 16S‐FL were significantly similar, showing both a comparable contrast between sites and a coherent sea‐land orientation within sites. In our dataset, 84.7% and 98.8% of the 16S‐V4V5 assigned reads were assigned strictly to the same species and genus, respectively, than those detected by 16S‐FL. 16S‐FL detected 92.2% of the 309 families and 87.7% of the 448 genera that were detected by the short 16S‐V4V5. 16S‐FL recorded 973 additional species and 392 genus not detected by 16S‐V4V5 (31.5% and 10.4% of the 16S‐FL reads, respectively, among which 67.8% and 79.3% were assigned), produced by both primer specificities and different error rates. Thus, our results concluded an overall similarity between 16S‐V4V5 and 16S‐FL sequencing strategies for this type of environmental samples.

Full genomic sequence characterization of the chikungunya virus from an imported case with serum viral concentration below culturable level

Chikungunya virus (CHIKV) infection, responsible for Chikungunya fever and occasionally severe symptoms, has emerged as an increasing global health concern following several large-scale outbreaks from Africa, Asia, Europe, and America. Over the past two decades, South and Southeast Asia regions have gradually become hot spots for outbreaks involving multiple CHIKV lineages. In China, most CHIKV infections are imported, making it crucial to trace the origins and transmission routes for effective prevention and control. In January 2024, a case of imported chikungunya fever was confirmed in Guangzhou City, Guangdong Province, China. However, the serum CHIKV viral concentration was too low for cultivation (RT-PCR detection, Ct = 32.62). Despite this, we successfully obtained the viral genome sequence directly from the whole blood sample using an optimized metatranscriptomic sequencing strategy, achieving a full-length viral genome with an average depth of 54.3X. Further analysis confirmed that the CHIKV virus belonged to the Asian lineage, traced to Timor-Leste, where an endemic CHIKV outbreak had been reported in January 2024, consistent with the patient’s travel history. Finally, we analyzed genetic evolutionary trends and amino acid site variations. This study highlights the identification of a CHIKV infection origin using direct whole-blood metatranscriptomic sequencing, a valuable method for rapidly sequencing low viral-load samples.

Targeted Therapies, Sequencing Strategies, and Beyond in Metastatic Hormone Receptor-Positive Breast Cancer: ASCO Guideline Clinical Insights.

Targeted Therapies, Sequencing Strategies, and Beyond in Metastatic Hormone Receptor-Positive Breast Cancer: ASCO Guideline Clinical Insights.

Next - generation sequencing whole genome respiratory syncytial virus in North Viet Nam, 2017 – 2020

Human Respiratory Syncytial Virus (RSV) infections pose a significant risk to both children and adults worldwide. 95% of children have been affected by RSV at least one time before 2 years old. This study aims to develop and apply a high-throughput sequencing strategy to sequence Vietnamese RSV whole genomes directly from clinical specimens in North Vietnam, 2017 - 2020. 38 clinical RSV-A and 43 clinical RSV-B samples were used to extract RNA, then converted to cDNA and PCR with specific primer sets. Next-generation sequencer with Illumina platform and analysis data by CLC Genomics Workbench. In silico analysis and laboratory testing revealed that the primer sets worked well and successfully amplified the whole genome of RSV. The nucleotide positions of gene start and gene end, as well as the substitutions in amino acid in each gene were determined. This protocol is simple to set up, easily scalable in sample preparation, and suitable for analysis. This reseach is the first public method of whole genome RSV sequencing and the most updated data of molecular RSV genome after 2015 in Viet Nam.