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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...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group
2016
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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. |
format | Online Article Text |
id | pubmed-4872057 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Nature Publishing Group |
record_format | MEDLINE/PubMed |
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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