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

• Published in: Scientific reports Vol. 6; no. 1; p. 26314
• Main Authors: Van den Hoecke, Silvie, Verhelst, Judith, Saelens, Xavier
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 19.05.2016; Nature Publishing Group
• Subjects: 45; 45/23; 631/208/457; 631/326; 631/326/596/1578; 64; Bias; DNA fragmentation; Gene expression; Genes, Reporter; Genetic diversity; Genetic Variation; Genome, Viral; Genomes; Genomics; Green fluorescent protein; Green Fluorescent Proteins - genetics; High-Throughput Nucleotide Sequencing - methods; Humanities and Social Sciences; Influenza; Influenza A; Influenza A virus - genetics; multidisciplinary; Nucleotide sequence; Polymerase chain reaction; Reporter gene; Reverse Transcriptase Polymerase Chain Reaction; Science; Sequence Analysis, DNA; Transposase; Viruses
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep26314

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.