BACKGROUND: DNA metabarcoding applies high-throughput sequencing approaches to generate numerous DNA barcodes from mixed sample pools for mass species identification and community characterisation. To date, however, most metabarcoding studies employ second-generation sequencing platforms like Illumina, which are limited by short read lengths and longer turnaround times. While third-generation platforms such as the MinION (Oxford Nanopore Technologies) can sequence longer reads and even in real-time, application of these platforms for metabarcoding has remained limited possibly due to the relatively high read error rates as well as the paucity of specialised software for processing such reads.
RESULTS: We show that this is no longer the case by performing nanopore-based, cytochrome c oxidase subunit I (COI) metabarcoding on 34 zooplankton bulk samples, and benchmarking the results against conventional Illumina MiSeq sequencing. Nanopore R10.3 sequencing chemistry and super accurate (SUP) basecalling model reduced raw read error rates to ~ 4%, and consensus calling with amplicon_sorter (without further error correction) generated metabarcodes that were ≤ 1% erroneous. Although Illumina recovered a higher number of molecular operational taxonomic units (MOTUs) than nanopore sequencing (589 vs. 471), we found no significant differences in the zooplankton communities inferred between the sequencing platforms. Importantly, 406 of 444 (91.4%) shared MOTUs between Illumina and nanopore were also found to be free of indel errors, and 85% of the zooplankton richness could be recovered after just 12-15 h of sequencing.
CONCLUSIONS: Our results demonstrate that nanopore sequencing can generate metabarcodes with Illumina-like accuracy, and we are the first study to show that nanopore metabarcodes are almost always indel-free. We also show that nanopore metabarcoding is viable for characterising species-rich communities rapidly, and that the same ecological conclusions can be obtained regardless of the sequencing platform used. Collectively, our study inspires confidence in nanopore sequencing and paves the way for greater utilisation of nanopore technology in various metabarcoding applications.

Multiple myeloma (MM) is the second most common hematologic malignant tumor. Standard holistic treatment model and new drug-based regimens have greatly improved the survival of patients with MM; however, minimal residual disease (MRD) causes relapse in most patients. Therefore, combining MRD testing on the basis of traditional serological efficacy evaluation is necessary to achieve a more accurate assessment of the patient\'s disease status. At present, next-generation flow cytometry (NGF) and next-generation sequencing (NGS) are the mainstream technologies for detecting MRD based on bone marrow samples. To standardize and normalize MRD detection, the expert group discussed and formulated Chinese expert consensus on the application of NGF and NGS technology for bone marrow MRD detection in patients with MM.
多发性骨髓瘤（MM）是血液系统第二大常见恶性肿瘤，标准的整体治疗模式及以新药为主的方案极大地改善了MM患者的生存，但微小残留病（MRD）导致大多数患者复发。因此，需要在传统血清学疗效评估的基础上联合MRD检测，以更精准地评估患者的疾病状态。目前，二代流式细胞术（NGF）和二代测序（NGS）是基于骨髓样本检测MRD的主流技术。为使MRD检测标准化和规范化，专家组讨论制订了在MM患者中应用NGF和NGS技术进行骨髓MRD检测的中国专家共识。.

Recently, high-throughput sequencing of influenza A viruses has become a routine test. It should be noted that the extremely high diversity of the influenza A virus complicates the task of determining the sequences of all eight genome segments. For a fast and accurate analysis, it is necessary to select the most suitable reference for each segment. At the same time, there is no standardized method in the field of decoding sequencing results that allows the user to update the sequence databases to which the reads obtained by virus sequencing are compared. The IAVCP (influenza A virus consensus and phylogeny) was developed with the goal of automatically analyzing high-throughput sequencing data of influenza A viruses. Its goals include the extraction of a consensus genome directly from paired raw reads. In addition, the pipeline enables the identification of potential reassortment events in the evolutionary history of the virus of interest by analyzing the topological structure of phylogenetic trees that are automatically reconstructed.

This study aimed to assess hematological diseases next-generation sequencing (NGS) panel enhances the diagnosis and classification of myeloid neoplasms (MN) using the 5th edition of the WHO Classification of Hematolymphoid Tumors (WHO-HAEM5) and the International Consensus Classification (ICC) of Myeloid Tumors. A cohort of 112 patients diagnosed with MN according to the revised fourth edition of the WHO classification (WHO-HAEM4R) underwent testing with a 141-gene NGS panel for hematological diseases. Ancillary studies were also conducted, including bone marrow cytomorphology and routine cytogenetics. The cases were then reclassified according to WHO-HAEM5 and ICC to assess the practical impact of these 2 classifications. The mutation detection rates were 93% for acute myeloid leukemia (AML), 89% for myelodysplastic syndrome (MDS), 94% for myeloproliferative neoplasm (MPN), and 100% for myelodysplasia/myeloproliferative neoplasm (MDS/MPN) (WHO-HAEM4R). NGS provided subclassified information for 26 and 29 patients with WHO-HAEM5 and ICC, respectively. In MPN, NGS confirmed diagnoses in 16 cases by detecting JAK2, MPL, or CALR mutations, whereas 13 \"triple-negative\" MPN cases revealed at least 1 mutation. NGS panel testing for hematological diseases improves the diagnosis and classification of MN. When diagnosed with ICC, NGS produces more classification subtype information than WHO-HAEM5.

BACKGROUND: The application of massive parallel sequencing technologies in the molecular analysis of Charcot-Marie-Tooth (CMT) has enabled the rapid and cost-effective identification of numerous potentially significant variants for diagnostic purposes. The objective is to reduce the number of variants, focusing only on those with pathogenic significance. The 2015 American College of Medical Genetics and Genomics (ACMG) guidelines aid in achieving this goal, but it is now evident that a pathology or gene-specific review of these rules is essential to avoid misinterpretations that may result from blindly applying the criteria. This study demonstrates how revised ACMG criteria, combined with CMT-specific literature data and expertise, can alter the final classification of a variant.
METHODS: We reviewed ACMG criteria based on current knowledge of CMT and provided suggestions for adapting them to the specificities of CMT.
RESULTS: Of the 226 index patients analysed, a diagnostic yield of 20% was obtained. It is worth noting that the 9% of cases had their final diagnosis changed with the application of the revised criteria, often resulting in the loss of the pathogenic classification of a variant.
CONCLUSIONS: The widespread availability of high-throughput sequencing technologies has enabled genetic testing even for laboratories without specific disease expertise. Disease-specific ACMG criteria can be a valuable tool to prevent the proliferation of variants of uncertain significance and the misinterpretation of variants.

OBJECTIVE: This document addresses the clinical application of next-generation sequencing (NGS) technologies for prenatal genetic diagnosis and aims to establish clinical practice recommendations in Spain to ensure uniformity in implementing these technologies into prenatal care.
METHODS: A joint committee of expert obstetricians and geneticists was created to review the existing literature on fetal NGS for genetic diagnosis and to make recommendations for Spanish healthcare professionals.
RESULTS: This guideline summarises technical aspects of NGS technologies, clinical indications in prenatal setting, considerations regarding findings to be reported, genetic counselling considerations as well as data storage and protection policies.
CONCLUSIONS: This document provides updated recommendations for the use of NGS diagnostic tests in prenatal diagnosis. These recommendations should be periodically reviewed as our knowledge of the clinical utility of NGS technologies, applied during pregnancy, may advance.

Fetal structural anomalies and birth defects are primarily caused by genetic variants such as chromosomal number abnormalities, copy number variations (CNV), single nucleotide variants (SNV), and small insertions and deletions (indel). Whole-genome sequencing (WGS) based on next-generation sequencing (NGS) as an emerging technology for genetic disease diagnosis can detect the aforementioned types of variants. In recent years, high-depth WGS (> 30×) for prenatal diagnosis has also become available, and proved to be practical for unraveling the genetic etiology of fetal developmental abnormalities. To facilitate clinical practice, test development and preliminary implementation of WGS for diagnosing fetal structural anomalies, we have formulated a consensus over the application of WGS in prenatal diagnosis by compiling previously published consensuses, guidelines, and research findings to provide a guidance on data analysis, reporting recommendations, and consultation of prenatal WGS results.

Enhancing the reproducibility and comprehension of adaptive immune receptor repertoire sequencing (AIRR-seq) data analysis is critical for scientific progress. This study presents guidelines for reproducible AIRR-seq data analysis, and a collection of ready-to-use pipelines with comprehensive documentation. To this end, ten common pipelines were implemented using ViaFoundry, a user-friendly interface for pipeline management and automation. This is accompanied by versioned containers, documentation and archiving capabilities. The automation of pre-processing analysis steps and the ability to modify pipeline parameters according to specific research needs are emphasized. AIRR-seq data analysis is highly sensitive to varying parameters and setups; using the guidelines presented here, the ability to reproduce previously published results is demonstrated. This work promotes transparency, reproducibility, and collaboration in AIRR-seq data analysis, serving as a model for handling and documenting bioinformatics pipelines in other research domains.

In hospital laboratories-developed testing is of great significance for the clinical testing products that has not been approved by the National Medical Product Administration and is urgently needed to meet clinical practice needs. With the development of cancer precision medicine in recent years, comprehensive genomic profiling (CGP) has become an important means and method for the detection of drug targets, precise molecular typing, and immunotherapy biomarkers in cancer patients. However, there is still a lack of unified understanding and consensus on clinical testing standards and application specifications for laboratory-developed testing in the hospitals. The Molecular Pathology Collaboration Group of the Cancer Experts Committee of the Chinese Anti-Cancer Association and the Molecular Pathology Group of the Pathology Branch of the Chinese Medical Association initiated the expert consensus on relevant specifications for analytical validation of CGP next-generation sequencing (NGS) testing in Chinese hospitals. Combined with domestic clinical practice, refer to domestic and foreign literatures, from the background of the laboratory-developed testing, analytical validation scenarios, evaluation indicators and variation ranges, sample types and quantities covered by analytical validation, clinical performance and drug efficacy determination, and site personnel for analytical validation, quality control, inter-laboratory quality evaluation and document management, etc. After the discussion by the expert group, 12 expert consensuses were formed to provide reference for the analytical validation and clinical application of tumor CGP NGS testing in Chinese hospitals, so as to promote the laboratory-developed testing applications in Chinese hospitals.
实验室自建检测对于医疗机构开展未经国家药品监督管理局审批且急需满足临床实践需求的检测具有重要意义。随着近年来肿瘤精准医学的发展，全景变异检测（CGP）已经成为肿瘤患者精准分子分型、靶向治疗、免疫治疗生物标志物等检测的重要手段和方法，但其在医疗机构开展自建检测的标准与应用规范尚缺乏统一的认识与共识。中国抗癌协会肿瘤病理专业委员会分子病理协作组联合中华医学会病理学分会分子病理学组发起了医疗机构开展CGP二代测序（NGS）检测性能确认相关规范中国专家共识，结合中国临床实践，参考国内外文献，从实验室自建的背景、性能确认的场景、评估指标与变异范围，性能确认覆盖的样本类型、数量、临床性能与药效判定，性能确认的场地、人员、质控、室间质评与文档管理等方面进行评述，经专家组讨论并形成12条专家共识，为中国医疗机构开展肿瘤CGP NGS检测的性能确认和临床应用提供参考，以促进医疗机构自建检测应用的发展。.

Metagenomic sequencing has emerged as a transformative tool in infectious disease diagnosis, offering a comprehensive and unbiased approach to pathogen detection. Leveraging international standards and guidelines is essential for ensuring the quality and reliability of metagenomic sequencing in clinical practice. This review explores the implications of international standards and guidelines for the application of metagenomic sequencing in infectious disease diagnosis. By adhering to established standards, such as those outlined by regulatory bodies and expert consensus, healthcare providers can enhance the accuracy and clinical utility of metagenomic sequencing. The integration of international standards and guidelines into metagenomic sequencing workflows can streamline diagnostic processes, improve pathogen identification, and optimize patient care. Strategies in implementing these standards for infectious disease diagnosis using metagenomic sequencing are discussed, highlighting the importance of standardized approaches in advancing precision infectious disease diagnosis initiatives.