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1.
Genome Biol ; 25(1): 176, 2024 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-38965568

RESUMO

Tandem repeats are frequent across the human genome, and variation in repeat length has been linked to a variety of traits. Recent improvements in long read sequencing technologies have the potential to greatly improve tandem repeat analysis, especially for long or complex repeats. Here, we introduce LongTR, which accurately genotypes tandem repeats from high-fidelity long reads available from both PacBio and Oxford Nanopore Technologies. LongTR is freely available at https://github.com/gymrek-lab/longtr and https://zenodo.org/doi/10.5281/zenodo.11403979 .


Assuntos
Variação Genética , Genoma Humano , Sequências de Repetição em Tandem , Humanos , Software , Análise de Sequência de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento por Nanoporos/métodos
2.
Nat Commun ; 15(1): 5580, 2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38961062

RESUMO

DNA methylation plays an important role in various biological processes, including cell differentiation, ageing, and cancer development. The most important methylation in mammals is 5-methylcytosine mostly occurring in the context of CpG dinucleotides. Sequencing methods such as whole-genome bisulfite sequencing successfully detect 5-methylcytosine DNA modifications. However, they suffer from the serious drawbacks of short read lengths and might introduce an amplification bias. Here we present Rockfish, a deep learning algorithm that significantly improves read-level 5-methylcytosine detection by using Nanopore sequencing. Rockfish is compared with other methods based on Nanopore sequencing on R9.4.1 and R10.4.1 datasets. There is an increase in the single-base accuracy and the F1 measure of up to 5 percentage points on R.9.4.1 datasets, and up to 0.82 percentage points on R10.4.1 datasets. Moreover, Rockfish shows a high correlation with whole-genome bisulfite sequencing, requires lower read depth, and achieves higher confidence in biologically important regions such as CpG-rich promoters while being computationally efficient. Its superior performance in human and mouse samples highlights its versatility for studying 5-methylcytosine methylation across varied organisms and diseases. Finally, its adaptable architecture ensures compatibility with new versions of pores and chemistry as well as modification types.


Assuntos
5-Metilcitosina , Ilhas de CpG , Metilação de DNA , Sequenciamento por Nanoporos , 5-Metilcitosina/metabolismo , 5-Metilcitosina/química , Sequenciamento por Nanoporos/métodos , Animais , Camundongos , Humanos , Ilhas de CpG/genética , Aprendizado Profundo , Algoritmos , Análise de Sequência de DNA/métodos , Sequenciamento Completo do Genoma/métodos , Sulfitos/química
3.
Genes Chromosomes Cancer ; 63(7): e23254, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38979775

RESUMO

An aneurysmal bone cyst (ABC) is a benign bone neoplasm that typically occurs during the first and second decades of life. ABC usually presents as a rapidly growing intramedullary expansile mass with multiple blood-filled cysts in the metaphysis of the long tubular bones. Here, we report a case of a periosteal solid ABC that was initially diagnosed as a high-grade surface osteosarcoma. A 10-year-old male was referred to our hospital for swelling and tenderness of the left upper arm. Radiography revealed periosteal mass without fluid-fluid levels. On performing open biopsy, the tumor showed hypercellular proliferation of uniform spindle to epithelioid cells with brisk mitotic activity (up to 12/2 mm2) and lace-like osteoid formation, which was diagnosed as a high-grade surface osteosarcoma. After one course of chemotherapy using adriamycin and cisplatin, peripheral sclerosis was conspicuous, which led to pathological review and revision of diagnosis as "possibly osteoblastoma." The patient was disease-free for 4 years after marginal resection and curettage. Retrospective nanopore DNA sequencing unexpectedly detected a PAFAH1B1::USP6 rearrangement. The fusion gene was further validated using reverse transcription-polymerase chain reaction and the diagnosis was revised to ABC. Chromothripsis involving chromosome 17 has also been identified. Methylation analysis classified the present tumor as an ABC or non-ossifying fibroma using t-distributed stochastic neighbor embedding and unsupervised hierarchical clustering. This case report highlights the utility of nanopore DNA sequencing for soft tissue and bone tumor diagnosis.


Assuntos
Cistos Ósseos Aneurismáticos , Cromotripsia , Sequenciamento por Nanoporos , Osteossarcoma , Ubiquitina Tiolesterase , Humanos , Masculino , Cistos Ósseos Aneurismáticos/genética , Cistos Ósseos Aneurismáticos/patologia , Cistos Ósseos Aneurismáticos/diagnóstico , Osteossarcoma/genética , Osteossarcoma/patologia , Osteossarcoma/diagnóstico , Ubiquitina Tiolesterase/genética , Criança , Sequenciamento por Nanoporos/métodos , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/diagnóstico , Rearranjo Gênico
4.
Microb Genom ; 10(7)2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38967541

RESUMO

Outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) are well described in the neonatal intensive care unit (NICU) setting. Genomics has revolutionized the investigation of such outbreaks; however, to date, this has largely been completed retrospectively and has typically relied on short-read platforms. In 2022, our laboratory established a prospective genomic surveillance system using Oxford Nanopore Technologies sequencing for rapid outbreak detection. Herein, using this system, we describe the detection and control of an outbreak of sequence-type (ST)97 MRSA in our NICU. The outbreak was identified 13 days after the first MRSA-positive culture and at a point where there were only two known cases. Ward screening rapidly defined the extent of the outbreak, with six other infants found to be colonized. There was minimal transmission once the outbreak had been detected and appropriate infection control measures had been instituted; only two further ST97 cases were detected, along with three unrelated non-ST97 MRSA cases. To contextualize the outbreak, core-genome single-nucleotide variants were identified for phylogenetic analysis after de novo assembly of nanopore data. Comparisons with global (n=45) and national surveillance (n=35) ST97 genomes revealed the stepwise evolution of methicillin resistance within this ST97 subset. A distinct cluster comprising nine of the ten ST97-IVa genomes from the NICU was identified, with strains from 2020 to 2022 national surveillance serving as outgroups to this cluster. One ST97-IVa genome presumed to be part of the outbreak formed an outgroup and was retrospectively excluded. A second phylogeny was created using Illumina sequencing, which considerably reduced the branch lengths of the NICU isolates on the phylogenetic tree. However, the overall tree topology and conclusions were unchanged, with the exception of the NICU outbreak cluster, where differences in branch lengths were observed. This analysis demonstrated the ability of a nanopore-only prospective genomic surveillance system to rapidly identify and contextualize an outbreak of MRSA in a NICU.


Assuntos
Surtos de Doenças , Unidades de Terapia Intensiva Neonatal , Staphylococcus aureus Resistente à Meticilina , Sequenciamento por Nanoporos , Filogenia , Infecções Estafilocócicas , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Staphylococcus aureus Resistente à Meticilina/classificação , Humanos , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Recém-Nascido , Sequenciamento por Nanoporos/métodos , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Estudos Prospectivos , Genoma Bacteriano , Polimorfismo de Nucleotídeo Único , Feminino
5.
BMC Infect Dis ; 24(1): 710, 2024 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-39030493

RESUMO

BACKGROUND: The clinical presentation of extrapulmonary tuberculosis (EPTB) is atypical and it is easily confused with other diseases such as common infections, making prompt diagnosis a great challenge. This study aimed to evaluate the accuracy of targeted nanopore sequencing (TNS) in the diagnosis of EPTB. The diagnostic accuracy of TNS using different types of extrapulmonary specimens was also evaluated. METHODS: We reviewed the clinical data of patients with suspected EPTB for whom TNS was conducted and who were hospitalized at our center. The true positive, false positive, false negative, and true negative values were determined. Indices of diagnostic accuracy were computed, including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the curve (AUC) for TNS and acid-fast bacilli (AFB) culture, and compared with those from clinical diagnosis. RESULTS: 149 patients were included in the analysis. The overall sensitivity, specificity, PPV, NPV, and AUC of TNS for the diagnosis of EPTB were 86.4%, 87.5%, 97.3%, 55.3%, and 0.87, respectively. For diagnosis by AFB culture, these values were 25.6%, 100.0%, 100.0%, 20.5%, and 0.63, respectively. The most common specimens used were lymph node tissue, cerebrospinal fluid, pleural effusion, and pleural tissue. The diagnostic accuracy of TNS using all types of extrapulmonary specimens was good. CONCLUSIONS: TNS demonstrates good diagnostic accuracy in the rapid diagnosis of EPTB and this was true across different types of extrapulmonary specimens.


Assuntos
Mycobacterium tuberculosis , Sequenciamento por Nanoporos , Sensibilidade e Especificidade , Tuberculose , Humanos , Tuberculose/diagnóstico , Tuberculose/microbiologia , Feminino , Masculino , Sequenciamento por Nanoporos/métodos , Pessoa de Meia-Idade , Adulto , Idoso , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Estudos Retrospectivos , Adulto Jovem , Valor Preditivo dos Testes , Tuberculose Extrapulmonar
6.
PLoS One ; 19(7): e0307389, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39024305

RESUMO

BACKGROUND: Rapid diagnosis of tuberculous meningitis (TBM) remains very difficult. Nanopore sequencing is gaining ground in the field of rapid tuberculosis (TB) diagnostics. The purpose of this study was to complete a protocol to guide the conduct of a systematic review and meta-analysis evaluating the accuracy of nanopore sequencing for the rapid diagnosis of TBM. METHODS: In accordance with the Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) guidelines, we completed this protocol, which was also registered on the PROSPERO platform. We will search the EMBASE, PubMed, the Cochrane Library, Wanfang database, and China National Knowledge Infrastructure databases for literature that evaluated the accuracy of nanopore sequencing for rapid diagnosis of TBM and screen them according to the inclusion and exclusion criteria, and qualified literature will be extracted with relevant data for further analysis. Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) will be used for evaluating the methodological quality of included studies. Stata (V 15.0; Stata Corp., College Station, TX, the USA) with midas module will be used to perform relevant meta-analysis. Heterogeneity between studies will be assessed by I2 statistics. When significant heterogeneity exists between studies, we will conduct meta-regression analyses, subgroup analyses and sensitivity analyses to further explore the sources of heterogeneity. CONCLUSION: We completed this study protocol, and this systematic review and meta-analysis will be the first systematic evaluation of the role of nanopore sequencing in the rapid diagnosis of TBM, which will allow clinicians to have a better understanding of the test. TRIAL REGISTRATION: Systematic review registration PROSPERO Registration number: CRD42024549837.


Assuntos
Metanálise como Assunto , Sequenciamento por Nanoporos , Revisões Sistemáticas como Assunto , Tuberculose Meníngea , Tuberculose Meníngea/diagnóstico , Tuberculose Meníngea/microbiologia , Humanos , Sequenciamento por Nanoporos/métodos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação
7.
BMC Genomics ; 25(1): 679, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38978005

RESUMO

BACKGROUND: Oxford Nanopore provides high throughput sequencing platforms able to reconstruct complete bacterial genomes with 99.95% accuracy. However, even small levels of error can obscure the phylogenetic relationships between closely related isolates. Polishing tools have been developed to correct these errors, but it is uncertain if they obtain the accuracy needed for the high-resolution source tracking of foodborne illness outbreaks. RESULTS: We tested 132 combinations of assembly and short- and long-read polishing tools to assess their accuracy for reconstructing the genome sequences of 15 highly similar Salmonella enterica serovar Newport isolates from a 2020 onion outbreak. While long-read polishing alone improved accuracy, near perfect accuracy (99.9999% accuracy or ~ 5 nucleotide errors across the 4.8 Mbp genome, excluding low confidence regions) was only obtained by pipelines that combined both long- and short-read polishing tools. Notably, medaka was a more accurate and efficient long-read polisher than Racon. Among short-read polishers, NextPolish showed the highest accuracy, but Pilon, Polypolish, and POLCA performed similarly. Among the 5 best performing pipelines, polishing with medaka followed by NextPolish was the most common combination. Importantly, the order of polishing tools mattered i.e., using less accurate tools after more accurate ones introduced errors. Indels in homopolymers and repetitive regions, where the short reads could not be uniquely mapped, remained the most challenging errors to correct. CONCLUSIONS: Short reads are still needed to correct errors in nanopore sequenced assemblies to obtain the accuracy required for source tracking investigations. Our granular assessment of the performance of the polishing pipelines allowed us to suggest best practices for tool users and areas for improvement for tool developers.


Assuntos
Benchmarking , Surtos de Doenças , Genoma Bacteriano , Nanoporos , Sequenciamento por Nanoporos/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Salmonella enterica/genética , Salmonella enterica/isolamento & purificação , Humanos , Filogenia
8.
PLoS One ; 19(6): e0304162, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38843269

RESUMO

BACKGROUND: Pulmonary tuberculosis (PTB) is the most common type of tuberculosis (TB). Rapid diagnosis of PTB can help in TB control. Although the use of molecular tests (such as the GeneXpert MTB/RIF) has improved the ability to rapidly diagnose PTB, there is still room for improvement. Nanopore sequencing is a novel means of rapid TB detection. The purpose of this study was to establish a systematic review and meta-analysis protocol for evaluating the accuracy of nanopore sequencing for the rapid diagnosis of PTB. METHODS: We completed this protocol according to the Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) statement and registered on the PROSPERO platform. We will screen studies related to nanopore sequencing for diagnosis of PTB by searching through PubMed, EMBASE, the Cochrane Library using English, and Wanfang database, CNKI (China National Knowledge Infrastructure) using Chinese. Eligible studies will be screened according to the inclusion and exclusion criteria established in the study protocol. We will evaluate the methodological quality of the individual included studies using Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2). We will use Stata (version 15.0) with the midas command and RevMan (version 5.3) for meta-analysis and forest plots and SROC curves generation. A p < 0.05 was treated as a statistically significant difference. When significant heterogeneity exists between studies, we will explore sources of heterogeneity through meta-regression analysis and subgroup analysis. CONCLUSION: To the best of our knowledge, this will be the first systematic review and meta-analysis of nanopore sequencing for the diagnosis of PTB. We hope that this study will find a new and effective tool for the early diagnosis of PTB. PROSPERO REGISTRATION NUMBER: CRD42023495593.


Assuntos
Metanálise como Assunto , Sequenciamento por Nanoporos , Revisões Sistemáticas como Assunto , Tuberculose Pulmonar , Tuberculose Pulmonar/diagnóstico , Humanos , Sequenciamento por Nanoporos/métodos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação
9.
BMJ Open ; 14(6): e080904, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38862231

RESUMO

OBJECTIVE: This study aimed to evaluate the efficiency of nanopore sequencing for the early diagnosis of tuberculous meningitis (TBM) using cerebrospinal fluid and compared it with acid-fast bacilli (AFB) smear, mycobacterial growth indicator tube culture and Xpert Mycobacterium tuberculosis (MTB)/rifampicin (RIF). DESIGN: Single-centre retrospective study. SETTING: The Tuberculosis Diagnosis and Treatment Center of Zhejiang Chinese and Western Medicine Integrated Hospital. PARTICIPANTS: We enrolled 64 adult patients with presumptive TBM admitted to our hospital from August 2021 to August 2023. METHODS: We calculated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of AFB smear, culture, Xpert MTB/RIF and nanopore sequencing to evaluate their diagnostic efficacy compared with a composite reference standard for TBM. RESULTS: Among these 64 patients, all tested negative for TBM by AFB smear. The sensitivity, specificity, PPV and NPV were 11.11%, 100%, 100% and 32.2% for culture, 13.33%, 100%, 100% and 2.76% for Xpert MTB/RIF, and 77.78%, 100%, 100% and 65.52% for nanopore sequencing, respectively. CONCLUSION: The diagnostic accuracy of the nanopore sequencing test was significantly higher than that of conventional testing methods used to detect TBM.


Assuntos
Mycobacterium tuberculosis , Sequenciamento por Nanoporos , Sensibilidade e Especificidade , Tuberculose Meníngea , Humanos , Tuberculose Meníngea/diagnóstico , Tuberculose Meníngea/líquido cefalorraquidiano , Tuberculose Meníngea/microbiologia , Estudos Retrospectivos , Masculino , Feminino , Adulto , China , Pessoa de Meia-Idade , Sequenciamento por Nanoporos/métodos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Valor Preditivo dos Testes , Idoso , Adulto Jovem , Líquido Cefalorraquidiano/microbiologia
10.
Nat Commun ; 15(1): 5148, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38890274

RESUMO

Telomere length is an important biomarker of organismal aging and cellular replicative potential, but existing measurement methods are limited in resolution and accuracy. Here, we deploy digital telomere measurement (DTM) by nanopore sequencing to understand how distributions of human telomere length change with age and disease. We measure telomere attrition and de novo elongation with up to 30 bp resolution in genetically defined populations of human cells, in blood cells from healthy donors and in blood cells from patients with genetic defects in telomere maintenance. We find that human aging is accompanied by a progressive loss of long telomeres and an accumulation of shorter telomeres. In patients with defects in telomere maintenance, the accumulation of short telomeres is more pronounced and correlates with phenotypic severity. We apply machine learning to train a binary classification model that distinguishes healthy individuals from those with telomere biology disorders. This sequencing and bioinformatic pipeline will advance our understanding of telomere maintenance mechanisms and the use of telomere length as a clinical biomarker of aging and disease.


Assuntos
Aprendizado de Máquina , Homeostase do Telômero , Telômero , Humanos , Telômero/genética , Telômero/metabolismo , Homeostase do Telômero/genética , Adulto , Envelhecimento Saudável/genética , Pessoa de Meia-Idade , Masculino , Idoso , Feminino , Encurtamento do Telômero/genética , Envelhecimento/genética , Sequenciamento por Nanoporos/métodos , Adulto Jovem
11.
Bioinformatics ; 40(6)2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38889266

RESUMO

MOTIVATION: Nanopore direct RNA sequencing (DRS) enables the detection of RNA N6-methyladenosine (m6A) without extra laboratory techniques. A number of supervised or comparative approaches have been developed to identify m6A from Nanopore DRS reads. However, existing methods typically utilize either statistical features of the current signals or basecalling-error features, ignoring the richer information of the raw signals of DRS reads. RESULTS: Here, we propose RedNano, a deep-learning method designed to detect m6A from Nanopore DRS reads by utilizing both raw signals and basecalling errors. RedNano processes the raw-signal feature and basecalling-error feature through residual networks. We validated the effectiveness of RedNano using synthesized, Arabidopsis, and human DRS data. The results demonstrate that RedNano surpasses existing methods by achieving higher area under the ROC curve (AUC) and area under the precision-recall curve (AUPRs) in all three datasets. Furthermore, RedNano performs better in cross-species validation, demonstrating its robustness. Additionally, when detecting m6A from an independent dataset of Populus trichocarpa, RedNano achieves the highest AUC and AUPR, which are 3.8%-9.9% and 5.5%-13.8% higher than other methods, respectively. AVAILABILITY AND IMPLEMENTATION: The source code of RedNano is freely available at https://github.com/Derryxu/RedNano.


Assuntos
Arabidopsis , Arabidopsis/genética , Humanos , Análise de Sequência de RNA/métodos , Adenosina/análogos & derivados , Adenosina/análise , Sequenciamento por Nanoporos/métodos , Aprendizado Profundo , RNA/química , Nanoporos
12.
Nat Commun ; 15(1): 5414, 2024 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-38926353

RESUMO

Borgs are huge extrachromosomal elements (ECE) of anaerobic methane-consuming "Candidatus Methanoperedens" archaea. Here, we used nanopore sequencing to validate published complete genomes curated from short reads and to reconstruct new genomes. 13 complete and four near-complete linear genomes share 40 genes that define a largely syntenous genome backbone. We use these conserved genes to identify new Borgs from peatland soil and to delineate Borg phylogeny, revealing two major clades. Remarkably, Borg genes encoding nanowire-like electron-transferring cytochromes and cell surface proteins are more highly expressed than those of host Methanoperedens, indicating that Borgs augment the Methanoperedens activity in situ. We reconstructed the first complete 4.00 Mbp genome for a Methanoperedens that is inferred to be a Borg host and predicted its methylation motifs, which differ from pervasive TC and CC methylation motifs of the Borgs. Thus, methylation may enable Methanoperedens to distinguish their genomes from those of Borgs. Very high Borg to Methanoperedens ratios and structural predictions suggest that Borgs may be capable of encapsulation. The findings clearly define Borgs as a distinct class of ECE with shared genomic signatures, establish their diversification from a common ancestor with genetic inheritance, and raise the possibility of periodic existence outside of host cells.


Assuntos
Genoma Arqueal , Metano , Filogenia , Metano/metabolismo , Oxirredução , Archaea/genética , Archaea/metabolismo , Sequenciamento por Nanoporos/métodos , Metilação de DNA , Microbiologia do Solo
13.
Genes (Basel) ; 15(6)2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38927663

RESUMO

Honeybees are an indispensable pollinator in nature with pivotal ecological, economic, and scientific value. However, a full-length transcriptome for Apis mellifera, assembled with the advanced third-generation nanopore sequencing technology, has yet to be reported. Here, nanopore sequencing of the midgut tissues of uninoculated and Nosema ceranae-inoculated A. mellifera workers was conducted, and the full-length transcriptome was then constructed and annotated based on high-quality long reads. Next followed improvement of sequences and annotations of the current reference genome of A. mellifera. A total of 5,942,745 and 6,664,923 raw reads were produced from midguts of workers at 7 days post-inoculation (dpi) with N. ceranae and 10 dpi, while 7,100,161 and 6,506,665 raw reads were generated from the midguts of corresponding uninoculated workers. After strict quality control, 6,928,170, 6,353,066, 5,745,048, and 6,416,987 clean reads were obtained, with a length distribution ranging from 1 kb to 10 kb. Additionally, 16,824, 17,708, 15,744, and 18,246 full-length transcripts were respectively detected, including 28,019 nonredundant ones. Among these, 43,666, 30,945, 41,771, 26,442, and 24,532 full-length transcripts could be annotated to the Nr, KOG, eggNOG, GO, and KEGG databases, respectively. Additionally, 501 novel genes (20,326 novel transcripts) were identified for the first time, among which 401 (20,255), 193 (13,365), 414 (19,186), 228 (12,093), and 202 (11,703) were respectively annotated to each of the aforementioned five databases. The expression and sequences of three randomly selected novel transcripts were confirmed by RT-PCR and Sanger sequencing. The 5' UTR of 2082 genes, the 3' UTR of 2029 genes, and both the 5' and 3' UTRs of 730 genes were extended. Moreover, 17,345 SSRs, 14,789 complete ORFs, 1224 long non-coding RNAs (lncRNAs), and 650 transcription factors (TFs) from 37 families were detected. Findings from this work not only refine the annotation of the A. mellifera reference genome, but also provide a valuable resource and basis for relevant molecular and -omics studies.


Assuntos
Anotação de Sequência Molecular , Transcriptoma , Abelhas/genética , Animais , Transcriptoma/genética , Genoma de Inseto , Nosema/genética , Sequenciamento por Nanoporos/métodos , Perfilação da Expressão Gênica/métodos
14.
Int J Mol Sci ; 25(12)2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38928350

RESUMO

The COVID-19 pandemic highlighted the need for a rapid, convenient, and scalable diagnostic method for detecting a novel pathogen amidst a global pandemic. While command-line interface tools offer automation for SARS-CoV-2 Oxford Nanopore Technology sequencing data analysis, they are inapplicable to users with limited programming skills. A solution is to establish such automated workflows within a graphical user interface software. We developed two workflows in the software Geneious Prime 2022.1.1, adapted for data obtained from the Midnight and Artic's nCoV-2019 sequencing protocols. Both workflows perform trimming, read mapping, consensus generation, and annotation on SARS-CoV-2 Nanopore sequencing data. Additionally, one workflow includes phylogenetic assignment using the bioinformatic tools pangolin and Nextclade as plugins. The basic workflow was validated in 2020, adhering to the requirements of the European Centre for Disease Prevention and Control for SARS-CoV-2 sequencing and analysis. The enhanced workflow, providing phylogenetic assignment, underwent validation at Uppsala University Hospital by analysing 96 clinical samples. It provided accurate diagnoses matching the original results of the basic workflow while also reducing manual clicks and analysis time. These bioinformatic workflows streamline SARS-CoV-2 Nanopore data analysis in Geneious Prime, saving time and manual work for operators lacking programming knowledge.


Assuntos
COVID-19 , Biologia Computacional , Pandemias , Filogenia , SARS-CoV-2 , Software , SARS-CoV-2/genética , COVID-19/epidemiologia , COVID-19/virologia , Humanos , Biologia Computacional/métodos , Fluxo de Trabalho , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Interface Usuário-Computador , Sequenciamento por Nanoporos/métodos
15.
Bioinformatics ; 40(Supplement_1): i287-i296, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38940135

RESUMO

SUMMARY: Improvements in nanopore sequencing necessitate efficient classification methods, including pre-filtering and adaptive sampling algorithms that enrich for reads of interest. Signal-based approaches circumvent the computational bottleneck of basecalling. But past methods for signal-based classification do not scale efficiently to large, repetitive references like pangenomes, limiting their utility to partial references or individual genomes. We introduce Sigmoni: a rapid, multiclass classification method based on the r-index that scales to references of hundreds of Gbps. Sigmoni quantizes nanopore signal into a discrete alphabet of picoamp ranges. It performs rapid, approximate matching using matching statistics, classifying reads based on distributions of picoamp matching statistics and co-linearity statistics, all in linear query time without the need for seed-chain-extend. Sigmoni is 10-100× faster than previous methods for adaptive sampling in host depletion experiments with improved accuracy, and can query reads against large microbial or human pangenomes. Sigmoni is the first signal-based tool to scale to a complete human genome and pangenome while remaining fast enough for adaptive sampling applications. AVAILABILITY AND IMPLEMENTATION: Sigmoni is implemented in Python, and is available open-source at https://github.com/vshiv18/sigmoni.


Assuntos
Algoritmos , Humanos , Sequenciamento por Nanoporos/métodos , Software , Nanoporos , Genoma Humano , Genômica/métodos , Análise de Sequência de DNA/métodos
16.
Viruses ; 16(6)2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38932133

RESUMO

Equine influenza is a viral disease caused by the equine influenza virus (EIV), and according to the WOAH, it is mandatory to report these infections. In Latin America and Colombia, EIV risk factors have not been analyzed. The objective of this research is to perform an epidemiological and molecular analysis of the EIV in horses with respiratory symptoms from 2020 to 2023 in Colombia. Molecular EIV detection was performed using RT-qPCR and nanopore sequencing. A risk analysis was also performed via the GEE method. A total of 188 equines with EIV respiratory symptoms were recruited. The positivity rate was 33.5%. The descriptive analysis showed that only 12.8% of the horses were vaccinated, and measures such as the quarantine and isolation of symptomatic animals accounted for 91.5% and 88.8%, respectively. The variables associated with the EIV were the non-isolation of positive individuals (OR = 8.16, 95% CI (1.52-43.67), p = 0.014) and sharing space with poultry (OR = 2.16, 95% CI (1.09-4.26), p = 0.027). In conclusion, this is the first EIV investigation in symptomatic horses in Colombia, highlighting the presence of the virus in the country and the need to improve preventive and control measures.


Assuntos
Doenças dos Cavalos , Vírus da Influenza A Subtipo H3N8 , Infecções por Orthomyxoviridae , Cavalos , Animais , Colômbia/epidemiologia , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/veterinária , Infecções por Orthomyxoviridae/virologia , Doenças dos Cavalos/virologia , Doenças dos Cavalos/epidemiologia , Vírus da Influenza A Subtipo H3N8/isolamento & purificação , Vírus da Influenza A Subtipo H3N8/genética , Feminino , Masculino , Filogenia , Sequenciamento por Nanoporos/métodos , Fatores de Risco
17.
Nat Commun ; 15(1): 5494, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38944650

RESUMO

Real-time genomics through nanopore sequencing holds the promise of fast antibiotic resistance prediction directly in the clinical setting. However, concerns about the accuracy of genomics-based resistance predictions persist, particularly when compared to traditional, clinically established diagnostic methods. Here, we leverage the case of a multi-drug resistant Klebsiella pneumoniae infection to demonstrate how real-time genomics can enhance the accuracy of antibiotic resistance profiling in complex infection scenarios. Our results show that unlike established diagnostics, nanopore sequencing data analysis can accurately detect low-abundance plasmid-mediated resistance, which often remains undetected by conventional methods. This capability has direct implications for clinical practice, where such "hidden" resistance profiles can critically influence treatment decisions. Consequently, the rapid, in situ application of real-time genomics holds significant promise for improving clinical decision-making and patient outcomes.


Assuntos
Antibacterianos , Farmacorresistência Bacteriana Múltipla , Genômica , Infecções por Klebsiella , Klebsiella pneumoniae , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/efeitos dos fármacos , Genômica/métodos , Humanos , Antibacterianos/farmacologia , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/diagnóstico , Farmacorresistência Bacteriana Múltipla/genética , Plasmídeos/genética , Sequenciamento por Nanoporos/métodos , Genoma Bacteriano/genética , Testes de Sensibilidade Microbiana
18.
Open Biol ; 14(6): 230449, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38862018

RESUMO

Nanopore sequencing platforms combined with supervised machine learning (ML) have been effective at detecting base modifications in DNA such as 5-methylcytosine (5mC) and N6-methyladenine (6mA). These ML-based nanopore callers have typically been trained on data that span all modifications on all possible DNA [Formula: see text]-mer backgrounds-a complete training dataset. However, as nanopore technology is pushed to more and more epigenetic modifications, such complete training data will not be feasible to obtain. Nanopore calling has historically been performed with hidden Markov models (HMMs) that cannot make successful calls for [Formula: see text]-mer contexts not seen during training because of their independent emission distributions. However, deep neural networks (DNNs), which share parameters across contexts, are increasingly being used as callers, often outperforming their HMM cousins. It stands to reason that a DNN approach should be able to better generalize to unseen [Formula: see text]-mer contexts. Indeed, herein we demonstrate that a common DNN approach (DeepSignal) outperforms a common HMM approach (Nanopolish) in the incomplete data setting. Furthermore, we propose a novel hybrid HMM-DNN approach, amortized-HMM, that outperforms both the pure HMM and DNN approaches on 5mC calling when the training data are incomplete. This type of approach is expected to be useful for calling other base modifications such as 5-hydroxymethylcytosine and for the simultaneous calling of different modifications, settings in which complete training data are not likely to be available.


Assuntos
5-Metilcitosina , Metilação de DNA , Epigênese Genética , Redes Neurais de Computação , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , 5-Metilcitosina/metabolismo , Sequenciamento por Nanoporos/métodos , Nanoporos , Humanos , Cadeias de Markov , DNA/química , DNA/genética
19.
Mol Cell ; 84(12): 2215-2217, 2024 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-38906112

RESUMO

In this issue, Li et al.1 report internal mRNA 2'-O-methyl (Nm) modification mapping by nanopore sequencing and the effect of Nm on mRNA stability and cancer cell progression.


Assuntos
Sequenciamento por Nanoporos , Neoplasias , Estabilidade de RNA , RNA Mensageiro , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequenciamento por Nanoporos/métodos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Metilação
20.
Microb Genom ; 10(6)2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38833287

RESUMO

It is now possible to assemble near-perfect bacterial genomes using Oxford Nanopore Technologies (ONT) long reads, but short-read polishing is usually required for perfection. However, the effect of short-read depth on polishing performance is not well understood. Here, we introduce Pypolca (with default and careful parameters) and Polypolish v0.6.0 (with a new careful parameter). We then show that: (1) all polishers other than Pypolca-careful, Polypolish-default and Polypolish-careful commonly introduce false-positive errors at low read depth; (2) most of the benefit of short-read polishing occurs by 25× depth; (3) Polypolish-careful almost never introduces false-positive errors at any depth; and (4) Pypolca-careful is the single most effective polisher. Overall, we recommend the following polishing strategies: Polypolish-careful alone when depth is very low (<5×), Polypolish-careful and Pypolca-careful when depth is low (5-25×), and Polypolish-default and Pypolca-careful when depth is sufficient (>25×).


Assuntos
Genoma Bacteriano , Nanoporos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Sequenciamento por Nanoporos/métodos , Bactérias/genética , Bactérias/classificação , Software , Genômica/métodos
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