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Importance of experimental information (metadata) for archived sequence data: case of specific gene bias due to lag time between sample harvest and RNA protection in RNA sequencing

Large volumes of high-throughput sequencing data have been submitted to the Sequencing Read Archive (SRA). The lack of experimental metadata associated with the data makes reuse and understanding data quality very difficult. In the case of RNA sequencing (RNA-Seq), which reveals the presence and qua...

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Autor principal: Matsuda, Tomoko
Formato: Online Artículo Texto
Lenguaje:English
Publicado: PeerJ Inc. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8401820/
https://www.ncbi.nlm.nih.gov/pubmed/34527435
http://dx.doi.org/10.7717/peerj.11875
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author Matsuda, Tomoko
author_facet Matsuda, Tomoko
author_sort Matsuda, Tomoko
collection PubMed
description Large volumes of high-throughput sequencing data have been submitted to the Sequencing Read Archive (SRA). The lack of experimental metadata associated with the data makes reuse and understanding data quality very difficult. In the case of RNA sequencing (RNA-Seq), which reveals the presence and quantity of RNA in a biological sample at any moment, it is necessary to consider that gene expression responds over a short time interval (several seconds to a few minutes) in many organisms. Therefore, to isolate RNA that accurately reflects the transcriptome at the point of harvest, raw biological samples should be processed by freezing in liquid nitrogen, immersing in RNA stabilization reagent or lysing and homogenizing in RNA lysis buffer containing guanidine thiocyanate as soon as possible. As the number of samples handled simultaneously increases, the time until the RNA is protected can increase. Here, to evaluate the effect of different lag times in RNA protection on RNA-Seq data, we harvested CHO-S cells after 3, 5, 6, and 7 days of cultivation, added RNA lysis buffer in a time course of 15, 30, 45, and 60 min after harvest, and conducted RNA-Seq. These RNA samples showed high RNA integrity number (RIN) values indicating non-degraded RNA, and sequence data from libraries prepared with these RNA samples was of high quality according to FastQC. We observed that, at the same cultivation day, global trends of gene expression were similar across the time course of addition of RNA lysis buffer; however, the expression of some genes was significantly different between the time-course samples of the same cultivation day; most of these differentially expressed genes were related to apoptosis. We conclude that the time lag between sample harvest and RNA protection influences gene expression of specific genes. It is, therefore, necessary to know not only RIN values of RNA and the quality of the sequence data but also how the experiment was performed when acquiring RNA-Seq data from the database.
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spelling pubmed-84018202021-09-14 Importance of experimental information (metadata) for archived sequence data: case of specific gene bias due to lag time between sample harvest and RNA protection in RNA sequencing Matsuda, Tomoko PeerJ Bioinformatics Large volumes of high-throughput sequencing data have been submitted to the Sequencing Read Archive (SRA). The lack of experimental metadata associated with the data makes reuse and understanding data quality very difficult. In the case of RNA sequencing (RNA-Seq), which reveals the presence and quantity of RNA in a biological sample at any moment, it is necessary to consider that gene expression responds over a short time interval (several seconds to a few minutes) in many organisms. Therefore, to isolate RNA that accurately reflects the transcriptome at the point of harvest, raw biological samples should be processed by freezing in liquid nitrogen, immersing in RNA stabilization reagent or lysing and homogenizing in RNA lysis buffer containing guanidine thiocyanate as soon as possible. As the number of samples handled simultaneously increases, the time until the RNA is protected can increase. Here, to evaluate the effect of different lag times in RNA protection on RNA-Seq data, we harvested CHO-S cells after 3, 5, 6, and 7 days of cultivation, added RNA lysis buffer in a time course of 15, 30, 45, and 60 min after harvest, and conducted RNA-Seq. These RNA samples showed high RNA integrity number (RIN) values indicating non-degraded RNA, and sequence data from libraries prepared with these RNA samples was of high quality according to FastQC. We observed that, at the same cultivation day, global trends of gene expression were similar across the time course of addition of RNA lysis buffer; however, the expression of some genes was significantly different between the time-course samples of the same cultivation day; most of these differentially expressed genes were related to apoptosis. We conclude that the time lag between sample harvest and RNA protection influences gene expression of specific genes. It is, therefore, necessary to know not only RIN values of RNA and the quality of the sequence data but also how the experiment was performed when acquiring RNA-Seq data from the database. PeerJ Inc. 2021-08-25 /pmc/articles/PMC8401820/ /pubmed/34527435 http://dx.doi.org/10.7717/peerj.11875 Text en © 2021 Matsuda https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.
spellingShingle Bioinformatics
Matsuda, Tomoko
Importance of experimental information (metadata) for archived sequence data: case of specific gene bias due to lag time between sample harvest and RNA protection in RNA sequencing
title Importance of experimental information (metadata) for archived sequence data: case of specific gene bias due to lag time between sample harvest and RNA protection in RNA sequencing
title_full Importance of experimental information (metadata) for archived sequence data: case of specific gene bias due to lag time between sample harvest and RNA protection in RNA sequencing
title_fullStr Importance of experimental information (metadata) for archived sequence data: case of specific gene bias due to lag time between sample harvest and RNA protection in RNA sequencing
title_full_unstemmed Importance of experimental information (metadata) for archived sequence data: case of specific gene bias due to lag time between sample harvest and RNA protection in RNA sequencing
title_short Importance of experimental information (metadata) for archived sequence data: case of specific gene bias due to lag time between sample harvest and RNA protection in RNA sequencing
title_sort importance of experimental information (metadata) for archived sequence data: case of specific gene bias due to lag time between sample harvest and rna protection in rna sequencing
topic Bioinformatics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8401820/
https://www.ncbi.nlm.nih.gov/pubmed/34527435
http://dx.doi.org/10.7717/peerj.11875
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