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Article: NanoRTax, a real-time pipeline for taxonomic and diversity analysis of nanopore 16S rRNA amplicon sequencing data.

Rodríguez-Pérez, Héctor / Ciuffreda, Laura / Flores, Carlos

Computational and structural biotechnology journal

2022 Volume 20, Page(s) 5350–5354

Abstract: Background: The study of microbial communities and their applications have been leveraged by advances in sequencing techniques and bioinformatics tools. The Oxford Nanopore Technologies long-read sequencing by nanopores provides a portable and cost- ... ...

Abstract	Background: The study of microbial communities and their applications have been leveraged by advances in sequencing techniques and bioinformatics tools. The Oxford Nanopore Technologies long-read sequencing by nanopores provides a portable and cost-efficient platform for sequencing assays. While this opens the possibility of sequencing applications outside specialized environments and real-time analysis of data, complementing the existing efficient library preparation protocols with streamlined bioinformatic workflows is required. Results: Here we present NanoRTax, a Nextflow pipeline for nanopore 16S rRNA gene amplicon data that features state-of-the-art taxonomic classification tools and real-time capability. The pipeline is paired with a web-based visual interface to enable user-friendly inspections of the experiment in progress. NanoRTax workflow and a simulated real-time analysis were used to validate the prediction of adult Intensive Care Unit patient mortality based on full-length 16S rRNA sequencing data from respiratory microbiome samples. Conclusions: This constitutes a proof-of-concept simulation study of how real-time bioinformatic workflows could be used to shorten the turnaround times in critical care settings and provides an instrument for future research on early-response strategies for sepsis.
Language	English
Publishing date	2022-09-23
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	2694435-2
ISSN	2001-0370
ISSN	2001-0370
DOI	10.1016/j.csbj.2022.09.024
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Article: Nanopore sequencing and its application to the study of microbial communities.

Ciuffreda, Laura / Rodríguez-Pérez, Héctor / Flores, Carlos

Computational and structural biotechnology journal

2021 Volume 19, Page(s) 1497–1511

Abstract: Since its introduction, nanopore sequencing has enhanced our ability to study complex microbial samples through the possibility to sequence long reads in real time using inexpensive and portable technologies. The use of long reads has allowed to address ... ...

Abstract	Since its introduction, nanopore sequencing has enhanced our ability to study complex microbial samples through the possibility to sequence long reads in real time using inexpensive and portable technologies. The use of long reads has allowed to address several previously unsolved issues in the field, such as the resolution of complex genomic structures, and facilitated the access to metagenome assembled genomes (MAGs). Furthermore, the low cost and portability of platforms together with the development of rapid protocols and analysis pipelines have featured nanopore technology as an attractive and ever-growing tool for real-time in-field sequencing for environmental microbial analysis. This review provides an up-to-date summary of the experimental protocols and bioinformatic tools for the study of microbial communities using nanopore sequencing, highlighting the most important and recent research in the field with a major focus on infectious diseases. An overview of the main approaches including targeted and shotgun approaches, metatranscriptomics, epigenomics, and epitranscriptomics is provided, together with an outlook to the major challenges and perspectives over the use of this technology for microbial studies.
Language	English
Publishing date	2021-03-07
Publishing country	Netherlands
Document type	Journal Article ; Review
ZDB-ID	2694435-2
ISSN	2001-0370
ISSN	2001-0370
DOI	10.1016/j.csbj.2021.02.020
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Article ; Online: NanoCLUST: a species-level analysis of 16S rRNA nanopore sequencing data.

Rodríguez-Pérez, Héctor / Ciuffreda, Laura / Flores, Carlos

Bioinformatics (Oxford, England)

2020 Volume 37, Issue 11, Page(s) 1600–1601

Abstract: Summary: NanoCLUST is an analysis pipeline for the classification of amplicon-based full-length 16S rRNA nanopore reads. It is characterized by an unsupervised read clustering step, based on Uniform Manifold Approximation and Projection (UMAP), followed ...

Abstract	Summary: NanoCLUST is an analysis pipeline for the classification of amplicon-based full-length 16S rRNA nanopore reads. It is characterized by an unsupervised read clustering step, based on Uniform Manifold Approximation and Projection (UMAP), followed by the construction of a polished read and subsequent Blast classification. Here, we demonstrate that NanoCLUST performs better than other state-of-the-art software in the characterization of two commercial mock communities, enabling accurate bacterial identification and abundance profile estimation at species-level resolution. Availability and implementation: Source code, test data and documentation of NanoCLUST are freely available at https://github.com/genomicsITER/NanoCLUST under MIT License. Supplementary information: Supplementary data are available at Bioinformatics online.
MeSH term(s)	High-Throughput Nucleotide Sequencing ; Nanopore Sequencing ; Nanopores ; RNA, Ribosomal, 16S/genetics ; Sequence Analysis, DNA ; Software
Chemical Substances	RNA, Ribosomal, 16S
Language	English
Publishing date	2020-10-20
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1422668-6
ISSN	1367-4811 ; 1367-4803
ISSN (online)	1367-4811
ISSN	1367-4803
DOI	10.1093/bioinformatics/btaa900
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Article ; Online: Nanopore sequencing and its application to the study of microbial communities

Ciuffreda, Laura / Rodríguez-Pérez, Héctor / Flores Villela, Carlos Arturo

Computational and Structural Biotechnology Journal. 2021, v. 19 p.1497-1511

2021

Abstract	Since its introduction, nanopore sequencing has enhanced our ability to study complex microbial samples through the possibility to sequence long reads in real time using inexpensive and portable technologies. The use of long reads has allowed to address several previously unsolved issues in the field, such as the resolution of complex genomic structures, and facilitated the access to metagenome assembled genomes (MAGs). Furthermore, the low cost and portability of platforms together with the development of rapid protocols and analysis pipelines have featured nanopore technology as an attractive and ever-growing tool for real-time in-field sequencing for environmental microbial analysis. This review provides an up-to-date summary of the experimental protocols and bioinformatic tools for the study of microbial communities using nanopore sequencing, highlighting the most important and recent research in the field with a major focus on infectious diseases. An overview of the main approaches including targeted and shotgun approaches, metatranscriptomics, epigenomics, and epitranscriptomics is provided, together with an outlook to the major challenges and perspectives over the use of this technology for microbial studies.
Keywords	bioinformatics ; biotechnology ; epigenetics ; genome ; metagenomics ; nanopores ; transcriptomics ; AMR ; HMW ; LCA ; MAGs ; NGS ; ONT ; PAIs ; SARS-CoV-2 ; UMAP ; Nanopore sequencing ; Targeted sequencing ; Metagenomics ; Metatranscriptomics ; Bioinformatics
Language	English
Size	p. 1497-1511.
Publishing place	Elsevier B.V.
Document type	Article ; Online
ZDB-ID	2694435-2
ISSN	2001-0370
ISSN	2001-0370
DOI	10.1016/j.csbj.2021.02.020
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: NanoRTax, a real-time pipeline for taxonomic and diversity analysis of nanopore 16S rRNA amplicon sequencing data

Rodríguez-Pérez, Héctor / Ciuffreda, Laura / Flores Villela, Carlos Arturo

Computational and Structural Biotechnology Journal. 2022, v. 20 p.5350-5354

2022

Abstract: The study of microbial communities and their applications have been leveraged by advances in sequencing techniques and bioinformatics tools. The Oxford Nanopore Technologies long-read sequencing by nanopores provides a portable and cost-efficient ... ...

Abstract	The study of microbial communities and their applications have been leveraged by advances in sequencing techniques and bioinformatics tools. The Oxford Nanopore Technologies long-read sequencing by nanopores provides a portable and cost-efficient platform for sequencing assays. While this opens the possibility of sequencing applications outside specialized environments and real-time analysis of data, complementing the existing efficient library preparation protocols with streamlined bioinformatic workflows is required. Here we present NanoRTax, a Nextflow pipeline for nanopore 16S rRNA gene amplicon data that features state-of-the-art taxonomic classification tools and real-time capability. The pipeline is paired with a web-based visual interface to enable user-friendly inspections of the experiment in progress. NanoRTax workflow and a simulated real-time analysis were used to validate the prediction of adult Intensive Care Unit patient mortality based on full-length 16S rRNA sequencing data from respiratory microbiome samples. This constitutes a proof-of-concept simulation study of how real-time bioinformatic workflows could be used to shorten the turnaround times in critical care settings and provides an instrument for future research on early-response strategies for sepsis.
Keywords	Internet ; adults ; bioinformatics ; biotechnology ; cost effectiveness ; genes ; microbiome ; mortality ; nanopores ; patients ; prediction ; NGS ; ONT ; ICU ; AUC ; ROC ; Pipeline ; Sequencing ; Nanopore ; Real-time ; Microbiome
Language	English
Size	p. 5350-5354.
Publishing place	Elsevier B.V.
Document type	Article ; Online
Note	Creative Commons Attribution 4.0 Generic (CC BY 4.0) ; Resource is Open Access
ZDB-ID	2694435-2
ISSN	2001-0370
ISSN	2001-0370
DOI	10.1016/j.csbj.2022.09.024
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Estimation of parasite age and synchrony status in Plasmodium falciparum infections.

Ciuffreda, Laura / Zoiku, Felix Kwame / Quashie, Neils B / Ranford-Cartwright, Lisa C

Scientific reports

2020 Volume 10, Issue 1, Page(s) 10925

Abstract: Human malaria parasites have complex but poorly understood population dynamics inside their human host. In some but not all infections, parasites progress synchronously through the 48 h lifecycle following erythrocyte invasion, such that at any one time ... ...

Abstract	Human malaria parasites have complex but poorly understood population dynamics inside their human host. In some but not all infections, parasites progress synchronously through the 48 h lifecycle following erythrocyte invasion, such that at any one time there is a limited spread of parasites at a particular time (hours) post-invasion. Patients presenting with older parasites, and with asynchronous infections, have been reported to have higher risks of fatal outcomes, associated with higher parasite biomass and multiplication rates respectively. However, practical tools to assess synchrony and estimate parasite age post-invasion in patient samples are lacking. We have developed a novel method based on three genes differentially expressed over the parasite intra-erythrocytic lifecycle, and applied it to samples from patients with uncomplicated malaria attending two health clinics in Ghana. We found that most patients presented with synchronous infections, and with parasites within 12 h of erythrocyte invasion. Finally we investigated if clinical features such as fever and parasite density could act as predictors of parasite age and synchrony. The new method is a simple and practicable approach to study parasite dynamics in naturally-infected patients, and is a significant improvement on the subjective microscopical methods for parasite staging in vivo, aiding patient management.
MeSH term(s)	Aging ; Animals ; Ethnic Groups ; Gene Expression Regulation, Developmental ; Ghana ; Humans ; Life Cycle Stages ; Malaria, Falciparum/parasitology ; Models, Biological ; Parasitemia/parasitology ; Plasmodium falciparum/genetics ; Plasmodium falciparum/growth & development ; Plasmodium falciparum/physiology
Language	English
Publishing date	2020-07-02
Publishing country	England
Document type	Journal Article ; Multicenter Study ; Research Support, Non-U.S. Gov't ; Validation Study
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-020-67817-6
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Article ; Online: Estimation of parasite age and synchrony status in Plasmodium falciparum infections

Laura Ciuffreda / Felix Kwame Zoiku / Neils B. Quashie / Lisa C. Ranford-Cartwright

Scientific Reports, Vol 10, Iss 1, Pp 1-

2020 Volume 10

Abstract: Abstract Human malaria parasites have complex but poorly understood population dynamics inside their human host. In some but not all infections, parasites progress synchronously through the 48 h lifecycle following erythrocyte invasion, such that at any ... ...

Abstract	Abstract Human malaria parasites have complex but poorly understood population dynamics inside their human host. In some but not all infections, parasites progress synchronously through the 48 h lifecycle following erythrocyte invasion, such that at any one time there is a limited spread of parasites at a particular time (hours) post-invasion. Patients presenting with older parasites, and with asynchronous infections, have been reported to have higher risks of fatal outcomes, associated with higher parasite biomass and multiplication rates respectively. However, practical tools to assess synchrony and estimate parasite age post-invasion in patient samples are lacking. We have developed a novel method based on three genes differentially expressed over the parasite intra-erythrocytic lifecycle, and applied it to samples from patients with uncomplicated malaria attending two health clinics in Ghana. We found that most patients presented with synchronous infections, and with parasites within 12 h of erythrocyte invasion. Finally we investigated if clinical features such as fever and parasite density could act as predictors of parasite age and synchrony. The new method is a simple and practicable approach to study parasite dynamics in naturally-infected patients, and is a significant improvement on the subjective microscopical methods for parasite staging in vivo, aiding patient management.
Keywords	Medicine ; R ; Science ; Q
Subject code	572 ; 616
Language	English
Publishing date	2020-07-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Reinfection rate and disease severity of the BA.5 Omicron SARS-CoV-2 lineage compared to previously circulating variants of concern in the Canary Islands (Spain).

Ciuffreda, Laura / Lorenzo-Salazar, José M / García-Martínez de Artola, Diego / Gil-Campesino, Helena / Alcoba-Florez, Julia / Rodríguez-Pérez, Héctor / Íñigo-Campos, Antonio / Salas-Hernández, Josmar / Rodríguez-Nuñez, Julia / Muñoz-Barrera, Adrián / Valenzuela-Fernández, Agustín / Díez-Gil, Oscar / González-Montelongo, Rafaela / Flores, Carlos

Emerging microbes & infections

2023 Volume 12, Issue 1, Page(s) 2202281

Abstract: ... ...

Abstract	ABSTRACT
MeSH term(s)	Humans ; SARS-CoV-2 ; Spain ; COVID-19 ; Reinfection ; Patient Acuity
Language	English
Publishing date	2023-05-04
Publishing country	United States
Document type	Observational Study ; Journal Article
ZDB-ID	2681359-2
ISSN	2222-1751 ; 2222-1751
ISSN (online)	2222-1751
ISSN	2222-1751
DOI	10.1080/22221751.2023.2202281
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Article ; Online: Association of the Delta SARS-CoV-2 variant with 28-day hospital mortality between December 2020 and September 2021.

Ciuffreda, Laura / Alcoba-Florez, Julia / Lorenzo-Salazar, José M / Gil-Campesino, Helena / García-Martínez de Artola, Diego / Díez-Gil, Oscar / Rodríguez-Pérez, Héctor / Íñigo-Campos, Antonio / Valenzuela-Fernández, Agustín / González-Montelongo, Rafaela / Flores, Carlos

The Journal of infection

2022 Volume 85, Issue 1, Page(s) 90–122

MeSH term(s)	COVID-19 ; Hospital Mortality ; Humans ; SARS-CoV-2
Language	English
Publishing date	2022-04-22
Publishing country	England
Document type	Letter ; Research Support, Non-U.S. Gov't
ZDB-ID	424417-5
ISSN	1532-2742 ; 0163-4453
ISSN (online)	1532-2742
ISSN	0163-4453
DOI	10.1016/j.jinf.2022.04.030
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Article ; Online: Sensitivity of different RT-qPCR solutions for SARS-CoV-2 detection.

Alcoba-Florez, Julia / Gil-Campesino, Helena / Artola, Diego García-Martínez de / González-Montelongo, Rafaela / Valenzuela-Fernández, Agustín / Ciuffreda, Laura / Flores, Carlos

International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases

2020 Volume 99, Page(s) 190–192

Abstract: Objectives: The ongoing COVID-19 pandemic continues to impose demands on diagnostic screening. In anticipation that the recurrence of outbreaks and the measures for lifting the lockdown worldwide may cause supply chain issues over the coming months, ... ...

Abstract	Objectives: The ongoing COVID-19 pandemic continues to impose demands on diagnostic screening. In anticipation that the recurrence of outbreaks and the measures for lifting the lockdown worldwide may cause supply chain issues over the coming months, this study assessed the sensitivity of a number of one-step retrotranscription and quantitative polymerase chain reaction (RT-qPCR) solutions to detect SARS-CoV-2. Methods: Six different RT-qPCR alternatives were evaluated for SARS-CoV-2/COVID-19 diagnosis based on standard RNA extractions. The one with best sensitivity was also assessed with direct nasopharyngeal swab viral transmission medium (VTM) heating; thus overcoming the RNA extraction step. Results: A wide variability in the sensitivity of RT-qPCR solutions was found that was associated with a range of false negatives from 2% (0.3-7.9%) to 39.8% (30.2-50.2%). Direct preheating of VTM combined with the best solution provided a sensitivity of 72.5% (62.5-81.0%), in the range of some of the solutions based on standard RNA extractions. Conclusions: Sensitivity limitations of currently used RT-qPCR solutions were found. These results will help to calibrate the impact of false negative diagnoses of COVID-19, and to detect and control new SARS-CoV-2 outbreaks and community transmissions.
MeSH term(s)	Betacoronavirus/genetics ; Betacoronavirus/isolation & purification ; COVID-19 ; Coronavirus Envelope Proteins ; Coronavirus Infections/diagnosis ; Coronavirus Infections/virology ; False Negative Reactions ; Humans ; Nasopharynx/virology ; Pandemics ; Pneumonia, Viral/diagnosis ; Pneumonia, Viral/virology ; Real-Time Polymerase Chain Reaction/methods ; SARS-CoV-2 ; Sensitivity and Specificity ; Viral Envelope Proteins/genetics
Chemical Substances	Coronavirus Envelope Proteins ; Viral Envelope Proteins ; envelope protein, SARS-CoV-2
Keywords	covid19
Language	English
Publishing date	2020-08-01
Publishing country	Canada
Document type	Journal Article
ZDB-ID	1331197-9
ISSN	1878-3511 ; 1201-9712
ISSN (online)	1878-3511
ISSN	1201-9712
DOI	10.1016/j.ijid.2020.07.058
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