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Evaluation of Oxford Nanopore MinION RNA-Seq Performance for Human Primary Cells

Transcript sequencing is a crucial tool for gaining a deep understanding of biological processes in diagnostic and clinical medicine. Given their potential to study novel complex eukaryotic transcriptomes, long-read sequencing technologies are able to overcome some limitations of short-read RNA-Seq...

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Autores principales: Massaiu, Ilaria, Songia, Paola, Chiesa, Mattia, Valerio, Vincenza, Moschetta, Donato, Alfieri, Valentina, Myasoedova, Veronika A., Schmid, Michael, Cassetta, Luca, Colombo, Gualtiero I., D’Alessandra, Yuri, Poggio, Paolo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8231517/
https://www.ncbi.nlm.nih.gov/pubmed/34204756
http://dx.doi.org/10.3390/ijms22126317
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author Massaiu, Ilaria
Songia, Paola
Chiesa, Mattia
Valerio, Vincenza
Moschetta, Donato
Alfieri, Valentina
Myasoedova, Veronika A.
Schmid, Michael
Cassetta, Luca
Colombo, Gualtiero I.
D’Alessandra, Yuri
Poggio, Paolo
author_facet Massaiu, Ilaria
Songia, Paola
Chiesa, Mattia
Valerio, Vincenza
Moschetta, Donato
Alfieri, Valentina
Myasoedova, Veronika A.
Schmid, Michael
Cassetta, Luca
Colombo, Gualtiero I.
D’Alessandra, Yuri
Poggio, Paolo
author_sort Massaiu, Ilaria
collection PubMed
description Transcript sequencing is a crucial tool for gaining a deep understanding of biological processes in diagnostic and clinical medicine. Given their potential to study novel complex eukaryotic transcriptomes, long-read sequencing technologies are able to overcome some limitations of short-read RNA-Seq approaches. Oxford Nanopore Technologies (ONT) offers the ability to generate long-read sequencing data in real time via portable protein nanopore USB devices. This work aimed to provide the user with the number of reads that should be sequenced, through the ONT MinION platform, to reach the desired accuracy level for a human cell RNA study. We sequenced three cDNA libraries prepared from poly-adenosine RNA of human primary cardiac fibroblasts. Since the runs were comparable, they were combined in a total dataset of 48 million reads. Synthetic datasets with different sizes were generated starting from the total and analyzed in terms of the number of identified genes and their expression levels. As expected, an improved sensitivity was obtained, increasing the sequencing depth, particularly for the non-coding genes. The reliability of expression levels was assayed by (i) comparison with PCR quantifications of selected genes and (ii) by the implementation of a user-friendly multiplexing method in a single run.
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spelling pubmed-82315172021-06-26 Evaluation of Oxford Nanopore MinION RNA-Seq Performance for Human Primary Cells Massaiu, Ilaria Songia, Paola Chiesa, Mattia Valerio, Vincenza Moschetta, Donato Alfieri, Valentina Myasoedova, Veronika A. Schmid, Michael Cassetta, Luca Colombo, Gualtiero I. D’Alessandra, Yuri Poggio, Paolo Int J Mol Sci Article Transcript sequencing is a crucial tool for gaining a deep understanding of biological processes in diagnostic and clinical medicine. Given their potential to study novel complex eukaryotic transcriptomes, long-read sequencing technologies are able to overcome some limitations of short-read RNA-Seq approaches. Oxford Nanopore Technologies (ONT) offers the ability to generate long-read sequencing data in real time via portable protein nanopore USB devices. This work aimed to provide the user with the number of reads that should be sequenced, through the ONT MinION platform, to reach the desired accuracy level for a human cell RNA study. We sequenced three cDNA libraries prepared from poly-adenosine RNA of human primary cardiac fibroblasts. Since the runs were comparable, they were combined in a total dataset of 48 million reads. Synthetic datasets with different sizes were generated starting from the total and analyzed in terms of the number of identified genes and their expression levels. As expected, an improved sensitivity was obtained, increasing the sequencing depth, particularly for the non-coding genes. The reliability of expression levels was assayed by (i) comparison with PCR quantifications of selected genes and (ii) by the implementation of a user-friendly multiplexing method in a single run. MDPI 2021-06-12 /pmc/articles/PMC8231517/ /pubmed/34204756 http://dx.doi.org/10.3390/ijms22126317 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Massaiu, Ilaria
Songia, Paola
Chiesa, Mattia
Valerio, Vincenza
Moschetta, Donato
Alfieri, Valentina
Myasoedova, Veronika A.
Schmid, Michael
Cassetta, Luca
Colombo, Gualtiero I.
D’Alessandra, Yuri
Poggio, Paolo
Evaluation of Oxford Nanopore MinION RNA-Seq Performance for Human Primary Cells
title Evaluation of Oxford Nanopore MinION RNA-Seq Performance for Human Primary Cells
title_full Evaluation of Oxford Nanopore MinION RNA-Seq Performance for Human Primary Cells
title_fullStr Evaluation of Oxford Nanopore MinION RNA-Seq Performance for Human Primary Cells
title_full_unstemmed Evaluation of Oxford Nanopore MinION RNA-Seq Performance for Human Primary Cells
title_short Evaluation of Oxford Nanopore MinION RNA-Seq Performance for Human Primary Cells
title_sort evaluation of oxford nanopore minion rna-seq performance for human primary cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8231517/
https://www.ncbi.nlm.nih.gov/pubmed/34204756
http://dx.doi.org/10.3390/ijms22126317
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