The complete genome sequence of unculturable Mycoplasma faucium obtained through clinical metagenomic next-generation sequencing.

Introduction: Diagnosing Mycoplasma faucium poses challenges, and it's unclear if its rare isolation is due to infrequent occurrence or its fastidious nutritional requirements. Methods: This study analyzes the complete genome sequence of M. faucium, obtained directly from the pus of a sternum infection in a lung transplant patient using metagenomic sequencing. Results: Genome analysis revealed limited therapeutic options for the M. faucium infection, primarily susceptibility to tetracyclines. Three classes of mobile genetic elements were identified: two new insertion sequences, a new prophage (phiUMCG-1), and a species-specific variant of a mycoplasma integrative and conjugative element (MICE). Additionally, a Type I Restriction-Modification system was identified, featuring 5'-terminally truncated hsdS pseudogenes with overlapping repeats, indicating the potential for forming alternative hsdS variants through recombination. Conclusion: This study represents the first-ever acquisition of a complete circularized bacterial genome directly from a patient sample obtained from invasive infection of a primary sterile site using culture-independent, PCR-free clinical metagenomics.

Rapid detection of the source of a Listeria monocytogenes outbreak in Switzerland through routine interviewing of patients and whole-genome sequencing.

AIMS OF THE STUDY: Listeriosis is a notifiable disease in Switzerland. In summer 2022, the Swiss Federal Office of Public Health noticed an increase in reports of listeriosis cases, indicating a possible ongoing outbreak. Here we present the approaches applied for rapidly confirming the outbreak, detecting the underlying source of infection and the measures put in place to eliminate it and contain the outbreak. METHODS: For close surveillance and early detection of outbreak situations with their possible sources, listeriosis patients in Switzerland are systematically interviewed about risk behaviours and foods consumed prior to the infection. Listeria monocytogenes isolates derived from patients in medical laboratories are sent to the National Reference Laboratory for Enteropathogenic Bacteria and Listeria, where they routinely undergo whole-genome sequencing. Interview and whole-genome sequencing data are continuously linked for comparison and analysis. RESULTS: In summer 2022, 20 patient-derived L. monocytogenes serotype 4b sequence type 388 strains were found to belong to an outbreak cluster (≤10 different alleles between neighbouring isolates) based on core genome multilocus sequence typing analysis. Geographically, 18 of 20 outbreak cases occurred in northeastern Switzerland. The median age of patients was 77.4 years (range: 58.1-89.7), with both sexes equally affected. Rolling analysis of the interview data revealed smoked trout from a local producer as a suspected infection source, triggering an on-site investigation of the production facility and sampling of the suspected products by the responsible cantonal food inspection team on 15 July 2022. Seven of ten samples tested positive for L. monocytogenes and the respective cantonal authority ordered a ban on production and distribution as well as a product recall. The Federal Food Safety and Veterinary Office released a nationwide public alert covering the smoked fish products concerned. Whole-genome sequencing analysis confirmed the interrelatedness of the L. monocytogenes smoked trout product isolates and the patient-derived isolates. Following the ban on production and distribution and the product recall, reporting of new outbreak-related cases rapidly dropped to zero. CONCLUSIONS: This listeriosis outbreak could be contained within a relatively short time thanks to identification of the source of contamination through the established combined approach of timely interviewing of every listeriosis patient or a representative and continuous molecular analysis of the patient- and food-derived L. monocytogenes isolates. These findings highlight the effectiveness of this well-established, joint approach involving the federal and cantonal authorities and the research institutions mandated to contain listeriosis outbreaks in Switzerland.

SNP-SVant: A Computational Workflow to Predict and Annotate Genomic Variants in Organisms Lacking Benchmarked Variants.

Whole-genome sequencing is widely used to investigate population genomic variation in organisms of interest. Assorted tools have been independently developed to call variants from short-read sequencing data aligned to a reference genome, including single nucleotide polymorphisms (SNPs) and structural variations (SVs). We developed SNP-SVant, an integrated, flexible, and computationally efficient bioinformatic workflow that predicts high-confidence SNPs and SVs in organisms without benchmarked variants, which are traditionally used for distinguishing sequencing errors from real variants. In the absence of these benchmarked datasets, we leverage multiple rounds of statistical recalibration to increase the precision of variant prediction. The SNP-SVant workflow is flexible, with user options to tradeoff accuracy for sensitivity. The workflow predicts SNPs and small insertions and deletions using the Genome Analysis ToolKit (GATK) and predicts SVs using the Genome Rearrangement IDentification Software Suite (GRIDSS), and it culminates in variant annotation using custom scripts. A key utility of SNP-SVant is its scalability. Variant calling is a computationally expensive procedure, and thus, SNP-SVant uses a workflow management system with intermediary checkpoint steps to ensure efficient use of resources by minimizing redundant computations and omitting steps where dependent files are available. SNP-SVant also provides metrics to assess the quality of called variants and converts between VCF and aligned FASTA format outputs to ensure compatibility with downstream tools to calculate selection statistics, which are commonplace in population genomics studies. By accounting for both small and large structural variants, users of this workflow can obtain a wide-ranging view of genomic alterations in an organism of interest. Overall, this workflow advances our capabilities in assessing the functional consequences of different types of genomic alterations, ultimately improving our ability to associate genotypes with phenotypes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Predicting single nucleotide polymorphisms and structural variations Support Protocol 1: Downloading publicly available sequencing data Support Protocol 2: Visualizing variant loci using Integrated Genome Viewer Support Protocol 3: Converting between VCF and aligned FASTA formats.

Whole-genome sequencing of Ganoderma boninense, the causal agent of basal stem rot disease in oil palm, via combined short- and long-read sequencing.

The hemibiotrophic Basidiomycete pathogen Ganoderma boninense (Gb) is the dominant causal agent of oil palm basal stem rot disease. Here, we report a complete chromosomal genome map of Gb using a combination of short-read Illumina and long-read Pacific Biosciences (PacBio) sequencing platforms combined with chromatin conformation capture data from the Chicago and Hi-C platforms. The genome was 55.87 Mb in length and assembled to a high contiguity (N50: 304.34 kb) of 12 chromosomes built from 112 scaffolds, with a total of only 4.34 Mb (~ 7.77%) remaining unplaced. The final assemblies were evaluated for completeness of the genome by using Benchmarking Universal Single Copy Orthologs (BUSCO) v4.1.4, and based on 4464 total BUSCO polyporales group searches, the assemblies yielded 4264 (95.52%) of the conserved orthologs as complete and only a few fragmented BUSCO of 42 (0.94%) as well as a missing BUSCO of 158 (3.53%). Genome annotation predicted a total of 21,074 coding genes, with a GC content ratio of 59.2%. The genome features were analyzed with different databases, which revealed 2471 Gene Ontology/GO (11.72%), 5418 KEGG (Kyoto Encyclopedia of Genes and Genomes) Orthologous/KO (25.71%), 13,913 Cluster of Orthologous Groups of proteins/COG (66.02%), 60 ABC transporter (0.28%), 1049 Carbohydrate-Active Enzymes/CAZy (4.98%), 4005 pathogen-host interactions/PHI (19%), and 515 fungal transcription factor/FTFD (2.44%) genes. The results obtained in this study provide deep insight for further studies in the future.

Evaluation of the accuracy of bacterial genome reconstruction with Oxford Nanopore R10.4.1 long-read-only sequencing.

Whole-genome reconstruction of bacterial pathogens has become an important tool for tracking transmission and antimicrobial resistance gene spread, but highly accurate and complete assemblies have largely only historically been achievable using hybrid long- and short-read sequencing. We previously found the Oxford Nanopore Technologies (ONT) R10.4/kit12 flowcell/chemistry produced improved assemblies over the R9.4.1/kit10 combination, however long-read only assemblies contained more errors compared to Illumina-ONT hybrid assemblies. ONT have since released an R10.4.1/kit14 flowcell/chemistry upgrade and recommended the use of Bovine Serum Albumin (BSA) during library preparation, both of which reportedly increase accuracy and yield. They have also released updated basecallers trained using native bacterial DNA containing methylation sites intended to fix systematic basecalling errors, including common adenosine (A) to guanine (G) and cytosine (C) to thymine (T) substitutions. To evaluate these improvements, we successfully sequenced four bacterial reference strains, namely Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, and nine genetically diverse E. coli bloodstream infection-associated isolates from different phylogroups and sequence types, both with and without BSA. These sequences were de novo assembled and compared against Illumina-corrected reference genomes. In this small evaluation of 13 isolates we found that nanopore long-read-only R10.4.1/kit 14 assemblies with updated basecallers trained using bacterial methylated DNA produce accurate assemblies with ≥40×depth, sufficient to be cost-effective compared with hybrid ONT/Illumina sequencing in our setting.

Recurrent neural network for predicting absence of heterozygosity from low pass WGS with ultra-low depth.

BACKGROUND: The absence of heterozygosity (AOH) is a kind of genomic change characterized by a long contiguous region of homozygous alleles in a chromosome, which may cause human genetic disorders. However, no method of low-pass whole genome sequencing (LP-WGS) has been reported for the detection of AOH in a low-pass setting of less than onefold. We developed a method, termed CNVseq-AOH, for predicting the absence of heterozygosity using LP-WGS with ultra-low sequencing data, which overcomes the sparse nature of typical LP-WGS data by combing population-based haplotype information, adjustable sliding windows, and recurrent neural network (RNN). We tested the feasibility of CNVseq-AOH for the detection of AOH in 409 cases (11 AOH regions for model training and 863 AOH regions for validation) from the 1000 Genomes Project (1KGP). AOH detection using CNVseq-AOH was also performed on 6 clinical cases with previously ascertained AOHs by whole exome sequencing (WES). RESULTS: Using SNP-based microarray results as reference (AOHs detected by CNVseq-AOH with at least a 50% overlap with the AOHs detected by chromosomal microarray analysis), 409 samples (863 AOH regions) in the 1KGP were used for concordant analysis. For 784 AOHs on autosomes and 79 AOHs on the X chromosome, CNVseq-AOH can predict AOHs with a concordant rate of 96.23% and 59.49% respectively based on the analysis of 0.1-fold LP-WGS data, which is far lower than the current standard in the field. Using 0.1-fold LP-WGS data, CNVseq-AOH revealed 5 additional AOHs (larger than 10 Mb in size) in the 409 samples. We further analyzed AOHs larger than 10 Mb, which is recommended for reporting the possibility of UPD. For the 291 AOH regions larger than 10 Mb, CNVseq-AOH can predict AOHs with a concordant rate of 99.66% with only 0.1-fold LP-WGS data. In the 6 clinical cases, CNVseq-AOH revealed all 15 known AOH regions. CONCLUSIONS: Here we reported a method for analyzing LP-WGS data to accurately identify regions of AOH, which possesses great potential to improve genetic testing of AOH.

A multiomic characterization of the leukemia cell line REH using short- and long-read sequencing.

The B-cell acute lymphoblastic leukemia (ALL) cell line REH, with the t(12;21) ETV6::RUNX1 translocation, is known to have a complex karyotype defined by a series of large-scale chromosomal rearrangements. Taken from a 15-yr-old at relapse, the cell line offers a practical model for the study of pediatric B-ALL. In recent years, short- and long-read DNA and RNA sequencing have emerged as a complement to karyotyping techniques in the resolution of structural variants in an oncological context. Here, we explore the integration of long-read PacBio and Oxford Nanopore whole-genome sequencing, IsoSeq RNA sequencing, and short-read Illumina sequencing to create a detailed genomic and transcriptomic characterization of the REH cell line. Whole-genome sequencing clarified the molecular traits of disrupted ALL-associated genes including CDKN2A, PAX5, BTG1, VPREB1, and TBL1XR1, as well as the glucocorticoid receptor NR3C1 Meanwhile, transcriptome sequencing identified seven fusion genes within the genomic breakpoints. Together, our extensive whole-genome investigation makes high-quality open-source data available to the leukemia genomics community.

Distributions of candidate vaccine Targets, virulence Factors, and resistance features of invasive group B Streptococcus using Whole-Genome Sequencing: A Multicenter, population-based surveillance study.

BACKGROUND: Group B Streptococcus (GBS) is a leading cause of morbidity and mortality in young infants worldwide. This study aimed to investigate candidate GBS vaccine targets, virulence factors, and antimicrobial resistance determinants. METHODS: We used whole-genome sequencing to characterize invasive GBS isolates from infants < 3 months of age obtained from a multicenter population-based study conducted from 2015 to 2021 in China. RESULTS: Overall, seven serotypes were detected from 278 GBS isolates, four (Ia, Ib, III, V) of which accounted for 97.8 %. We detected 30 sequence types (including 10 novel types) that were grouped into six clonal complexes (CCs: CC1, CC10, CC17, CC19, CC23 and CC651); three novel ST groups in CC17 were detected, and the rate of CC17, considered a hyperinvasive neonatal clone complex, was attached to 40.6 % (113/278). A total of 98.9 % (275/278) of isolates harbored at least one alpha-like protein gene. All GBS isolates contained at least one of three pilus backbone determinants and the pilus types PI-2b and PI-1 + PI-2a accounted for 79.8 % of the isolates. The 112 serotype III/CC17 GBS isolates were all positive for hvgA. Most of the isolates (75.2 %) were positive for serine-rich repeat glycoprotein determinants (srr1or srr2). Almost all isolates possessed cfb (99.6 %), c1IE (100 %), lmb (95.3 %) or pavA (100 %) gene. Seventy-seven percent of isolates harboured more than three antimicrobial resistance genes with 28.4 % (79/278) gyrA quinoloneresistancedeterminants mutation, 83.8 % (233/278) carrying tet cluster genes and 77.3 % (215/278) carrying erm genes which mediated fluoroquinolone, tetracycline and clindamycin resistance, respectively." CONCLUSIONS: The findings from this large whole-genome sequence of GBS isolates establish important baseline data required for further surveillance and evaluating the impact of future vaccine candidates.

Single-cell mtDNA dynamics in tumors is driven by coregulation of nuclear and mitochondrial genomes.

The extent of cell-to-cell variation in tumor mitochondrial DNA (mtDNA) copy number and genotype, and the phenotypic and evolutionary consequences of such variation, are poorly characterized. Here we use amplification-free single-cell whole-genome sequencing (Direct Library Prep (DLP+)) to simultaneously assay mtDNA copy number and nuclear DNA (nuDNA) in 72,275 single cells derived from immortalized cell lines, patient-derived xenografts and primary human tumors. Cells typically contained thousands of mtDNA copies, but variation in mtDNA copy number was extensive and strongly associated with cell size. Pervasive whole-genome doubling events in nuDNA associated with stoichiometrically balanced adaptations in mtDNA copy number, implying that mtDNA-to-nuDNA ratio, rather than mtDNA copy number itself, mediated downstream phenotypes. Finally, multimodal analysis of DLP+ and single-cell RNA sequencing identified both somatic loss-of-function and germline noncoding variants in mtDNA linked to heteroplasmy-dependent changes in mtDNA copy number and mitochondrial transcription, revealing phenotypic adaptations to disrupted nuclear/mitochondrial balance.

DengueSeq: a pan-serotype whole genome amplicon sequencing protocol for dengue virus.

BACKGROUND: The increasing burden of dengue virus on public health due to more explosive and frequent outbreaks highlights the need for improved surveillance and control. Genomic surveillance of dengue virus not only provides important insights into the emergence and spread of genetically diverse serotypes and genotypes, but it is also critical to monitor the effectiveness of newly implemented control strategies. Here, we present DengueSeq, an amplicon sequencing protocol, which enables whole-genome sequencing of all four dengue virus serotypes. RESULTS: We developed primer schemes for the four dengue virus serotypes, which can be combined into a pan-serotype approach. We validated both approaches using genetically diverse virus stocks and clinical specimens that contained a range of virus copies. High genome coverage (>95%) was achieved for all genotypes, except DENV2 (genotype VI) and DENV 4 (genotype IV) sylvatics, with similar performance of the serotype-specific and pan-serotype approaches. The limit of detection to reach 70% coverage was 10-100 RNA copies/µL for all four serotypes, which is similar to other commonly used primer schemes. DengueSeq facilitates the sequencing of samples without known serotypes, allows the detection of multiple serotypes in the same sample, and can be used with a variety of library prep kits and sequencing instruments. CONCLUSIONS: DengueSeq was systematically evaluated with virus stocks and clinical specimens spanning the genetic diversity within each of the four dengue virus serotypes. The primer schemes can be plugged into existing amplicon sequencing workflows to facilitate the global need for expanded dengue virus genomic surveillance.

Whole-genome sequences restore the original classification of dabbling ducks (genus Anas).

Anas, is a genus of dabbling ducks and encompasses a considerable number of species, among which some are the progenitors of domestic ducks. However, the taxonomic position of the Anas genus remains uncertain because several of its species, initially categorized as Anas based on morphological characteristics, were subsequently reclassified and grouped with the South American genus Tachyeres, primarily based on analysis of their mitochondrial gene sequences. Here, we constructed a phylogenetic tree using nine of our recently assembled Anas genomes, two Tachyeres genomes, and one Cairina genome that are publicly available. The results showed that the Northern shoveler (Anas clypeata) and Baikal teal (Anas formosa) clustered with the other Anas species at the whole-genome level rather than with the Steamer ducks (genus Tachyeres). Therefore, we propose to restore the original classification of the Anas genus, which includes the Northern shoveler and Baikal teal species, 47 species in total. Moreover, our study unveiled extensive incomplete lineage sorting and an ancient introgression event from Tachyeres to Anas, which has led to notable phylogenetic incongruence within the Anas genome. This ancient introgression event not only supports the theory that Anas originated in South America but also that it played a significant role in shaping the evolutionary trajectory of Anas, including the domestic duck.

Whole Genome-Sequencing and Phylogenetic Analysis of Bacillus anthracis from 2019-2023 in Ningxia Hui Autonomous Region, China.

BACKGROUND: Since 2019, the incidence of anthrax in the Ningxia Hui Autonomous Region has increased significantly compared with previous years, so in this situation the anthrax in the Ningxia region not only had a detrimental impact on public health, but also inflicted significant economic repercussions. Therefore, we conducted a molecular epidemiological study of 20 strains from 2019-2023 isolates. This study investigated the origin of Bacillus anthracis and its genetic diversity. METHODS: We conducted canonical single-nucleotide polymorphisms (CanSNPs) typing and whole genome sequencing based on the extracted nucleic acid of Bacillus anthracis. Based on the whole genome drafts, we studied the genomic characteristics of 20 isolates. Meanwhile, we performed phylogenetic studies based on genome-wide core single-nucleotide polymorphisms (SNPs) using MEGA's Maximum Likelihood (ML) method and core-genome-based multilocus sequence typing (cgMLST) of the core genomes of these strains using BioNumerics' minimum spanning tree (MST) model. RESULTS: The 20 isolates were categorized into sub-lineages A.Br.001/002, and comparative genomic analyses of these strains with other isolates from other parts of the world showed that the strains from Ningxia were correlated with isolates from Europe, Indonesia, Georgia (USA), and Beijing (China). For the 20 isolates in Ningxia, the genetic relationship of the isolates isolated from the same year or region was relatively close. CONCLUSION: The A.Br.001/002 subgroup was the dominant endemic strain in Ningxia. The genetic relationship and phylogenesis between isolates from Ningxia and strains from Europe and Indonesia suggest that anthrax spread around the globe through ancient trade routes.

Krisp: A Python package to aid in the design of CRISPR and amplification-based diagnostic assays from whole genome sequencing data.

Recent pandemics like COVID-19 highlighted the importance of rapidly developing diagnostics to detect evolving pathogens. CRISPR-Cas technology has recently been used to develop diagnostic assays for sequence-specific recognition of DNA or RNA. These assays have similar sensitivity to the gold standard qPCR but can be deployed as easy to use and inexpensive test strips. However, the discovery of diagnostic regions of a genome flanked by conserved regions where primers can be designed requires extensive bioinformatic analyses of genome sequences. We developed the Python package krisp to aid in the discovery of primers and diagnostic sequences that differentiate groups of samples from each other, using either unaligned genome sequences or a variant call format (VCF) file as input. Krisp has been optimized to handle large datasets by using efficient algorithms that run in near linear time, use minimal RAM, and leverage parallel processing when available. The validity of krisp results has been demonstrated in the laboratory with the successful design of a CRISPR diagnostic assay to distinguish the sudden oak death pathogen Phytophthora ramorum from closely related Phytophthora species. Krisp is released open source under a permissive license with all the documentation needed to quickly design CRISPR-Cas diagnostic assays.

MicroPredict: predicting species-level taxonomic abundance of whole-shotgun metagenomic data using only 16S amplicon sequencing data.

BACKGROUND: The importance of the human microbiome in the analysis of various diseases is emerging. The two main methods used to profile the human microbiome are 16S rRNA gene sequencing (16S sequencing) and whole-genome shotgun sequencing (WGS). Owing to the full coverage of the genome in sequencing, WGS has multiple advantages over 16S sequencing, including higher taxonomic profiling resolution at the species-level and functional profiling analysis. However, 16S sequencing remains widely used because of its relatively low cost. Although WGS is the standard method for obtaining accurate species-level data, we found that 16S sequencing data contained rich information to predict high-resolution species-level abundances with reasonable accuracy. OBJECTIVE: In this study, we proposed MicroPredict, a method for accurately predicting WGS-comparable species-level abundance data using 16S taxonomic profile data. METHODS: We employed a mixed model using two key strategies: (1) modeling both sample- and species-specific information for predicting WGS abundances, and (2) accounting for the possible correlations among different species. RESULTS: We found that MicroPredict outperformed the other machine learning methods. CONCLUSION: We expect that our approach will help researchers accurately approximate the species-level abundances of microbiome profiles in datasets for which only cost-effective 16S sequencing has been applied.

Multimodal analysis of cfDNA methylomes for early detecting esophageal squamous cell carcinoma and precancerous lesions.

Detecting early-stage esophageal squamous cell carcinoma (ESCC) and precancerous lesions is critical for improving survival. Here, we conduct whole-genome bisulfite sequencing (WGBS) on 460 cfDNA samples from patients with non-metastatic ESCC or precancerous lesions and matched healthy controls. We develop an expanded multimodal analysis (EMMA) framework to simultaneously identify cfDNA methylation, copy number variants (CNVs), and fragmentation markers in cfDNA WGBS data. cfDNA methylation markers are the earliest and most sensitive, detectable in 70% of ESCCs and 50% of precancerous lesions, and associated with molecular subtypes and tumor microenvironments. CNVs and fragmentation features show high specificity but are linked to late-stage disease. EMMA significantly improves detection rates, increasing AUCs from 0.90 to 0.99, and detects 87% of ESCCs and 62% of precancerous lesions with >95% specificity in validation cohorts. Our findings demonstrate the potential of multimodal analysis of cfDNA methylome for early detection and monitoring of molecular characteristics in ESCC.

A solution to achieve sequencing from SARS-CoV-2 specimens with low viral loads: concatenation of reads from independent reactions.

BACKGROUND: During the pandemic, whole genome sequencing was critical to characterize SARS-CoV-2 for surveillance, clinical and therapeutical purposes. However, low viral loads in specimens often led to suboptimal sequencing, making lineage assignment and phylogenetic analysis difficult. We propose an alternative approach to sequencing these specimens that involves sequencing in triplicate and concatenation of the reads obtained using bioinformatics. This proposal is based on the hypothesis that the uncovered regions in each replicate differ and that concatenation would compensate for these gaps and recover a larger percentage of the sequenced genome. RESULTS: Whole genome sequencing was performed in triplicate on 30 samples with Ct > 32 and the benefit of replicate read concatenation was assessed. After concatenation: i) 28% of samples reached the standard quality coverage threshold (> 90% genome covered > 30x); ii) 39% of samples did not reach the coverage quality thresholds but coverage improved by more than 40%; and iii) SARS-CoV-2 lineage assignment was possible in 68.7% of samples where it had been impaired. CONCLUSIONS: Concatenation of reads from replicate sequencing reactions provides a simple way to access hidden information in the large proportion of SARS-CoV-2-positive specimens eliminated from analysis in standard sequencing schemes. This approach will enhance our potential to rule out involvement in outbreaks, to characterize reinfections and to identify lineages of concern for surveillance or therapeutical purposes.

vSNP: a SNP pipeline for the generation of transparent SNP matrices and phylogenetic trees from whole genome sequencing data sets.

BACKGROUND: Several single nucleotide polymorphism (SNP) pipelines exist, each offering its own advantages. Among them and described here is vSNP that has been developed over the past decade and is specifically tailored to meet the needs of diagnostic laboratories. Laboratories that aim to provide rapid whole genome sequencing results during outbreak investigations face unique challenges. vSNP addresses these challenges by enabling users to verify and validate sequence accuracy with ease- having utility across various pathogens, being fully auditable, and presenting results that are easy to interpret and can be comprehended by individuals with diverse backgrounds. RESULTS: vSNP has proven effective for real-time phylogenetic analysis of disease outbreaks and eradication efforts, including bovine tuberculosis, brucellosis, virulent Newcastle disease, SARS-CoV-2, African swine fever, and highly pathogenic avian influenza. The pipeline produces easy-to-read SNP matrices, sorted for convenience, as well as corresponding phylogenetic trees, making the output easily understandable. Essential data for verifying SNPs is included in the output, and the process has been divided into two steps for ease of use and faster processing times. vSNP requires minimal computational resources to run and can be run in a wide range of environments. Several utilities have been developed to make analysis more accessible for subject matter experts who may not have computational expertise. CONCLUSION: The vSNP pipeline integrates seamlessly into a diagnostic workflow and meets the criteria for quality control accreditation programs, such as 17025 by the International Organization for Standardization. Its versatility and robustness make it suitable for use with a diverse range of organisms, providing detailed, reproducible, and transparent results, making it a valuable tool in various applications, including phylogenetic analysis performed in real time.

[The updates of the ACMG variant interpretation guidelines affect the pathogenicity determination of OTOF gene variations in patients with auditory neuropathy].

Objective: To compare the differences between the variation interpretation standards and guidelines issued by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) in 2015 (The 2015ACMG/AMP guideline) and the Deafness Specialist Group of the Clinical Genome Resource (ClinGen) in 2018 for hereditary hearing loss (Healing loss, HL) issued the expert specification of the variation interpretation guide (The 2018 HL-EP guideline) in evaluating the pathogenicity of OTOF gene variation in patients with auditory neuropathy. Methods: Thirty-eight auditory neuropathy patients with OTOF gene variant were selected as the study subjects (23 males and 15 females, aged 0.3-25.9 years). Using whole-genome sequencing, whole exome sequencing or target region sequencing (Panel) combined with Sanger sequencing, 38 cases were found to carry more than two OTOF mutation sites. A total of 59 candidate variants were independently interpreted based on the 2015 ACMG/AMP guideline and 2018 HL-EP guideline. Compared with the judgment results in 2015 ACMG/AMP guideline, the variants interpreted as lower pathogenic classifications in the 2018 HL-EP guideline were defined as downgraded variants, and the variants regarded as higher pathogenic classifications were defined as upgraded variants. Statistical analysis was conducted using SPSS 20.0. Results: The concordance rate of variant classification between the guidelines was 72.9%(43/59). The 13.6%(8/59) of variants were upgraded and 13.6% (8/59) of variants downgraded in the classifications of the 2018 HL-EP guideline. A couple of rules saw significant differences between the guidelines (PVS1, PM3, PP2, PP3 and PP5). The distribution of pathogenicity of splicing mutation was statistically different (P=0.013). Conclusions: The 2018 HL-EP guideline is inconsistent with the 2015 ACMG/AMP guideline, when judging the pathogenicity of OTOF gene variants in patients with auditory neuropathy. Through the deletion and refinement of evidence and the breaking of solidification thinking, the 2018 HL-EP guideline makes the pathogenicity grading more traceable and improves the credibility.

[Expert consensus on the test development and preliminary implementation of whole genome sequencing for fetal structural abnormalities].

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.