title: High-Throughput, Amplicon-Based Sequencing of the CREBBP Gene as a Tool to Develop a Universal Platform-Independent Assay.
abstract: High-throughput sequencing technologies are widely used to analyse genomic variants or rare mutational events in different fields of genomic research, with a fast development of new or adapted platforms and technologies, enabling amplicon-based analysis of single target genes or even whole genome sequencing within a short period of time. Each sequencing platform is characterized by well-defined types of errors, resulting from different steps in the sequencing workflow. Here we describe a universal method to prepare amplicon libraries that can be used for sequencing on different high-throughput sequencing platforms. We have sequenced distinct exons of the CREB binding protein (CREBBP) gene and analysed the output resulting from three major deep-sequencing platforms. platform-specific errors were adjusted according to the result of sequence analysis from the remaining platforms. Additionally, bioinformatic methods are described to determine platform dependent errors. Summarizing the results we present a platform-independent cost-efficient and timesaving method that can be used as an alternative to commercially available sample-preparation kits.
authors.affiliation: 2nd Medical Department, University Hospital Schleswig-Holstein, Kiel, Germany.
authors.forename: Marc W
authors.initials: MW
authors.lastname: Fuellgrabe
authors.affiliation: 2nd Medical Department, University Hospital Schleswig-Holstein, Kiel, Germany.
authors.forename: Dietrich
authors.initials: D
authors.lastname: Herrmann
authors.affiliation: 2nd Medical Department, University Hospital Schleswig-Holstein, Kiel, Germany.
authors.forename: Henrik
authors.initials: H
authors.lastname: Knecht
authors.affiliation: Max-Planck Institute for Evolutionary Biology, Ploen, Germany.
authors.forename: Sven
authors.initials: S
authors.lastname: Kuenzel
authors.affiliation: 2nd Medical Department, University Hospital Schleswig-Holstein, Kiel, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Kneba
authors.affiliation: 2nd Medical Department, University Hospital Schleswig-Holstein, Kiel, Germany.
authors.forename: Christiane
authors.initials: C
authors.lastname: Pott
authors.affiliation: 2nd Medical Department, University Hospital Schleswig-Holstein, Kiel, Germany.
authors.forename: Monika
authors.initials: M
authors.lastname: Brüggemann
mesh_terms: D050882:CREB-Binding Protein / Q000235:genetics*; D019295:Computational Biology / Q000379:methods; D005091:Exons / Q000235:genetics; D015894:Genome, Human / Q000235:genetics*; D023281:Genomics / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans
chemical_list: D050882:CREB-Binding Protein; C494363:CREBBP protein, human