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Illumina MiSeq sequencing disfavours a sequence motif in the GFP reporter gene

Green fluorescent protein (GFP) is one of the most used reporter genes. We have used next-generation sequencing (NGS) to analyse the genetic diversity of a recombinant influenza A virus that expresses GFP and found a remarkable coverage dip in the GFP coding sequence. This coverage dip was present w...

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Autores principales: Van den Hoecke, Silvie, Verhelst, Judith, Saelens, Xavier
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872057/
https://www.ncbi.nlm.nih.gov/pubmed/27193250
http://dx.doi.org/10.1038/srep26314
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author Van den Hoecke, Silvie
Verhelst, Judith
Saelens, Xavier
author_facet Van den Hoecke, Silvie
Verhelst, Judith
Saelens, Xavier
author_sort Van den Hoecke, Silvie
collection PubMed
description Green fluorescent protein (GFP) is one of the most used reporter genes. We have used next-generation sequencing (NGS) to analyse the genetic diversity of a recombinant influenza A virus that expresses GFP and found a remarkable coverage dip in the GFP coding sequence. This coverage dip was present when virus-derived RT-PCR product or the parental plasmid DNA was used as starting material for NGS and regardless of whether Nextera XT transposase or Covaris shearing was used for DNA fragmentation. Therefore, the sequence coverage dip in the GFP coding sequence was not the result of emerging GFP mutant viruses or a bias introduced by Nextera XT fragmentation. Instead, we found that the Illumina MiSeq sequencing method disfavours the ‘CCCGCC’ motif in the GFP coding sequence.
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spelling pubmed-48720572016-06-01 Illumina MiSeq sequencing disfavours a sequence motif in the GFP reporter gene Van den Hoecke, Silvie Verhelst, Judith Saelens, Xavier Sci Rep Article Green fluorescent protein (GFP) is one of the most used reporter genes. We have used next-generation sequencing (NGS) to analyse the genetic diversity of a recombinant influenza A virus that expresses GFP and found a remarkable coverage dip in the GFP coding sequence. This coverage dip was present when virus-derived RT-PCR product or the parental plasmid DNA was used as starting material for NGS and regardless of whether Nextera XT transposase or Covaris shearing was used for DNA fragmentation. Therefore, the sequence coverage dip in the GFP coding sequence was not the result of emerging GFP mutant viruses or a bias introduced by Nextera XT fragmentation. Instead, we found that the Illumina MiSeq sequencing method disfavours the ‘CCCGCC’ motif in the GFP coding sequence. Nature Publishing Group 2016-05-19 /pmc/articles/PMC4872057/ /pubmed/27193250 http://dx.doi.org/10.1038/srep26314 Text en Copyright © 2016, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Van den Hoecke, Silvie
Verhelst, Judith
Saelens, Xavier
Illumina MiSeq sequencing disfavours a sequence motif in the GFP reporter gene
title Illumina MiSeq sequencing disfavours a sequence motif in the GFP reporter gene
title_full Illumina MiSeq sequencing disfavours a sequence motif in the GFP reporter gene
title_fullStr Illumina MiSeq sequencing disfavours a sequence motif in the GFP reporter gene
title_full_unstemmed Illumina MiSeq sequencing disfavours a sequence motif in the GFP reporter gene
title_short Illumina MiSeq sequencing disfavours a sequence motif in the GFP reporter gene
title_sort illumina miseq sequencing disfavours a sequence motif in the gfp reporter gene
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872057/
https://www.ncbi.nlm.nih.gov/pubmed/27193250
http://dx.doi.org/10.1038/srep26314
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