Consistent errors in first strand cDNA due to random hexamer mispriming.

Priming of random hexamers in cDNA synthesis is known to show sequence bias, but in addition it has been suggested recently that mismatches in random hexamer priming could be a cause of mismatches between the original RNA fragment and observed sequence reads. To explore random hexamer mispriming as...

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Autores principales:	Thomas P van Gurp, Lauren M McIntyre, Koen J F Verhoeven
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/0a0c8dd6777e43fca56e993bbfe3b81e
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spelling	oai:doaj.org-article:0a0c8dd6777e43fca56e993bbfe3b81e2021-11-18T08:39:43ZConsistent errors in first strand cDNA due to random hexamer mispriming.1932-620310.1371/journal.pone.0085583https://doaj.org/article/0a0c8dd6777e43fca56e993bbfe3b81e2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24386481/?tool=EBIhttps://doaj.org/toc/1932-6203Priming of random hexamers in cDNA synthesis is known to show sequence bias, but in addition it has been suggested recently that mismatches in random hexamer priming could be a cause of mismatches between the original RNA fragment and observed sequence reads. To explore random hexamer mispriming as a potential source of these errors, we analyzed two independently generated RNA-seq datasets of synthetic ERCC spikes for which the reference is known. First strand cDNA synthesized by random hexamer priming on RNA showed consistent position and nucleotide-specific mismatch errors in the first seven nucleotides. The mismatch errors found in both datasets are consistent in distribution and thermodynamically stable mismatches are more common. This strongly indicates that RNA-DNA mispriming of specific random hexamers causes these errors. Due to their consistency and specificity, mispriming errors can have profound implications for downstream applications if not dealt with properly.Thomas P van GurpLauren M McIntyreKoen J F VerhoevenPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 12, p e85583 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Thomas P van Gurp Lauren M McIntyre Koen J F Verhoeven Consistent errors in first strand cDNA due to random hexamer mispriming.
description	Priming of random hexamers in cDNA synthesis is known to show sequence bias, but in addition it has been suggested recently that mismatches in random hexamer priming could be a cause of mismatches between the original RNA fragment and observed sequence reads. To explore random hexamer mispriming as a potential source of these errors, we analyzed two independently generated RNA-seq datasets of synthetic ERCC spikes for which the reference is known. First strand cDNA synthesized by random hexamer priming on RNA showed consistent position and nucleotide-specific mismatch errors in the first seven nucleotides. The mismatch errors found in both datasets are consistent in distribution and thermodynamically stable mismatches are more common. This strongly indicates that RNA-DNA mispriming of specific random hexamers causes these errors. Due to their consistency and specificity, mispriming errors can have profound implications for downstream applications if not dealt with properly.
format	article
author	Thomas P van Gurp Lauren M McIntyre Koen J F Verhoeven
author_facet	Thomas P van Gurp Lauren M McIntyre Koen J F Verhoeven
author_sort	Thomas P van Gurp
title	Consistent errors in first strand cDNA due to random hexamer mispriming.
title_short	Consistent errors in first strand cDNA due to random hexamer mispriming.
title_full	Consistent errors in first strand cDNA due to random hexamer mispriming.
title_fullStr	Consistent errors in first strand cDNA due to random hexamer mispriming.
title_full_unstemmed	Consistent errors in first strand cDNA due to random hexamer mispriming.
title_sort	consistent errors in first strand cdna due to random hexamer mispriming.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/0a0c8dd6777e43fca56e993bbfe3b81e
work_keys_str_mv	AT thomaspvangurp consistenterrorsinfirststrandcdnaduetorandomhexamermispriming AT laurenmmcintyre consistenterrorsinfirststrandcdnaduetorandomhexamermispriming AT koenjfverhoeven consistenterrorsinfirststrandcdnaduetorandomhexamermispriming
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Consistent errors in first strand cDNA due to random hexamer mispriming.

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