Automated workflow for preparation of cDNA for cap analysis of gene expression on a single molecule sequencer.

<h4>Background</h4>Cap analysis of gene expression (CAGE) is a 5' sequence tag technology to globally determine transcriptional starting sites in the genome and their expression levels and has most recently been adapted to the HeliScope single molecule sequencer. Despite significant...

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Autores principales:	Masayoshi Itoh, Miki Kojima, Sayaka Nagao-Sato, Eri Saijo, Timo Lassmann, Mutsumi Kanamori-Katayama, Ai Kaiho, Marina Lizio, Hideya Kawaji, Piero Carninci, Alistair R R Forrest, Yoshihide Hayashizaki
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/812f9b1b98994c8f9777da73842d64fa
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spelling	oai:doaj.org-article:812f9b1b98994c8f9777da73842d64fa2021-11-18T07:29:07ZAutomated workflow for preparation of cDNA for cap analysis of gene expression on a single molecule sequencer.1932-620310.1371/journal.pone.0030809https://doaj.org/article/812f9b1b98994c8f9777da73842d64fa2012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22303458/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Cap analysis of gene expression (CAGE) is a 5' sequence tag technology to globally determine transcriptional starting sites in the genome and their expression levels and has most recently been adapted to the HeliScope single molecule sequencer. Despite significant simplifications in the CAGE protocol, it has until now been a labour intensive protocol.<h4>Methodology</h4>In this study we set out to adapt the protocol to a robotic workflow, which would increase throughput and reduce handling. The automated CAGE cDNA preparation system we present here can prepare 96 'HeliScope ready' CAGE cDNA libraries in 8 days, as opposed to 6 weeks by a manual operator.We compare the results obtained using the same RNA in manual libraries and across multiple automation batches to assess reproducibility.<h4>Conclusions</h4>We show that the sequencing was highly reproducible and comparable to manual libraries with an 8 fold increase in productivity. The automated CAGE cDNA preparation system can prepare 96 CAGE sequencing samples simultaneously. Finally we discuss how the system could be used for CAGE on Illumina/SOLiD platforms, RNA-seq and full-length cDNA generation.Masayoshi ItohMiki KojimaSayaka Nagao-SatoEri SaijoTimo LassmannMutsumi Kanamori-KatayamaAi KaihoMarina LizioHideya KawajiPiero CarninciAlistair R R ForrestYoshihide HayashizakiPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 1, p e30809 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Masayoshi Itoh Miki Kojima Sayaka Nagao-Sato Eri Saijo Timo Lassmann Mutsumi Kanamori-Katayama Ai Kaiho Marina Lizio Hideya Kawaji Piero Carninci Alistair R R Forrest Yoshihide Hayashizaki Automated workflow for preparation of cDNA for cap analysis of gene expression on a single molecule sequencer.
description	<h4>Background</h4>Cap analysis of gene expression (CAGE) is a 5' sequence tag technology to globally determine transcriptional starting sites in the genome and their expression levels and has most recently been adapted to the HeliScope single molecule sequencer. Despite significant simplifications in the CAGE protocol, it has until now been a labour intensive protocol.<h4>Methodology</h4>In this study we set out to adapt the protocol to a robotic workflow, which would increase throughput and reduce handling. The automated CAGE cDNA preparation system we present here can prepare 96 'HeliScope ready' CAGE cDNA libraries in 8 days, as opposed to 6 weeks by a manual operator.We compare the results obtained using the same RNA in manual libraries and across multiple automation batches to assess reproducibility.<h4>Conclusions</h4>We show that the sequencing was highly reproducible and comparable to manual libraries with an 8 fold increase in productivity. The automated CAGE cDNA preparation system can prepare 96 CAGE sequencing samples simultaneously. Finally we discuss how the system could be used for CAGE on Illumina/SOLiD platforms, RNA-seq and full-length cDNA generation.
format	article
author	Masayoshi Itoh Miki Kojima Sayaka Nagao-Sato Eri Saijo Timo Lassmann Mutsumi Kanamori-Katayama Ai Kaiho Marina Lizio Hideya Kawaji Piero Carninci Alistair R R Forrest Yoshihide Hayashizaki
author_facet	Masayoshi Itoh Miki Kojima Sayaka Nagao-Sato Eri Saijo Timo Lassmann Mutsumi Kanamori-Katayama Ai Kaiho Marina Lizio Hideya Kawaji Piero Carninci Alistair R R Forrest Yoshihide Hayashizaki
author_sort	Masayoshi Itoh
title	Automated workflow for preparation of cDNA for cap analysis of gene expression on a single molecule sequencer.
title_short	Automated workflow for preparation of cDNA for cap analysis of gene expression on a single molecule sequencer.
title_full	Automated workflow for preparation of cDNA for cap analysis of gene expression on a single molecule sequencer.
title_fullStr	Automated workflow for preparation of cDNA for cap analysis of gene expression on a single molecule sequencer.
title_full_unstemmed	Automated workflow for preparation of cDNA for cap analysis of gene expression on a single molecule sequencer.
title_sort	automated workflow for preparation of cdna for cap analysis of gene expression on a single molecule sequencer.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/812f9b1b98994c8f9777da73842d64fa
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Automated workflow for preparation of cDNA for cap analysis of gene expression on a single molecule sequencer.

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