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Suppression of artifacts and barcode bias in high-throughput transcriptome analyses utilizing template switching

Template switching (TS) has been an inherent mechanism of reverse transcriptase, which has been exploited in several transcriptome analysis methods, such as CAGE, RNA-Seq and short RNA sequencing. TS is an attractive option, given the simplicity of the protocol, which does not require an adaptor med...

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Autores principales: Tang, Dave T. P., Plessy, Charles, Salimullah, Md, Suzuki, Ana Maria, Calligaris, Raffaella, Gustincich, Stefano, Carninci, Piero
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562004/
https://www.ncbi.nlm.nih.gov/pubmed/23180801
http://dx.doi.org/10.1093/nar/gks1128
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author Tang, Dave T. P.
Plessy, Charles
Salimullah, Md
Suzuki, Ana Maria
Calligaris, Raffaella
Gustincich, Stefano
Carninci, Piero
author_facet Tang, Dave T. P.
Plessy, Charles
Salimullah, Md
Suzuki, Ana Maria
Calligaris, Raffaella
Gustincich, Stefano
Carninci, Piero
author_sort Tang, Dave T. P.
collection PubMed
description Template switching (TS) has been an inherent mechanism of reverse transcriptase, which has been exploited in several transcriptome analysis methods, such as CAGE, RNA-Seq and short RNA sequencing. TS is an attractive option, given the simplicity of the protocol, which does not require an adaptor mediated step and thus minimizes sample loss. As such, it has been used in several studies that deal with limited amounts of RNA, such as in single cell studies. Additionally, TS has also been used to introduce DNA barcodes or indexes into different samples, cells or molecules. This labeling allows one to pool several samples into one sequencing flow cell, increasing the data throughput of sequencing and takes advantage of the increasing throughput of current sequences. Here, we report TS artifacts that form owing to a process called strand invasion. Due to the way in which barcodes/indexes are introduced by TS, strand invasion becomes more problematic by introducing unsystematic biases. We describe a strategy that eliminates these artifacts in silico and propose an experimental solution that suppresses biases from TS.
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spelling pubmed-35620042013-02-01 Suppression of artifacts and barcode bias in high-throughput transcriptome analyses utilizing template switching Tang, Dave T. P. Plessy, Charles Salimullah, Md Suzuki, Ana Maria Calligaris, Raffaella Gustincich, Stefano Carninci, Piero Nucleic Acids Res Methods Online Template switching (TS) has been an inherent mechanism of reverse transcriptase, which has been exploited in several transcriptome analysis methods, such as CAGE, RNA-Seq and short RNA sequencing. TS is an attractive option, given the simplicity of the protocol, which does not require an adaptor mediated step and thus minimizes sample loss. As such, it has been used in several studies that deal with limited amounts of RNA, such as in single cell studies. Additionally, TS has also been used to introduce DNA barcodes or indexes into different samples, cells or molecules. This labeling allows one to pool several samples into one sequencing flow cell, increasing the data throughput of sequencing and takes advantage of the increasing throughput of current sequences. Here, we report TS artifacts that form owing to a process called strand invasion. Due to the way in which barcodes/indexes are introduced by TS, strand invasion becomes more problematic by introducing unsystematic biases. We describe a strategy that eliminates these artifacts in silico and propose an experimental solution that suppresses biases from TS. Oxford University Press 2013-02 2012-11-23 /pmc/articles/PMC3562004/ /pubmed/23180801 http://dx.doi.org/10.1093/nar/gks1128 Text en © The Author(s) 2012. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com.
spellingShingle Methods Online
Tang, Dave T. P.
Plessy, Charles
Salimullah, Md
Suzuki, Ana Maria
Calligaris, Raffaella
Gustincich, Stefano
Carninci, Piero
Suppression of artifacts and barcode bias in high-throughput transcriptome analyses utilizing template switching
title Suppression of artifacts and barcode bias in high-throughput transcriptome analyses utilizing template switching
title_full Suppression of artifacts and barcode bias in high-throughput transcriptome analyses utilizing template switching
title_fullStr Suppression of artifacts and barcode bias in high-throughput transcriptome analyses utilizing template switching
title_full_unstemmed Suppression of artifacts and barcode bias in high-throughput transcriptome analyses utilizing template switching
title_short Suppression of artifacts and barcode bias in high-throughput transcriptome analyses utilizing template switching
title_sort suppression of artifacts and barcode bias in high-throughput transcriptome analyses utilizing template switching
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562004/
https://www.ncbi.nlm.nih.gov/pubmed/23180801
http://dx.doi.org/10.1093/nar/gks1128
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