High-Throughput, Amplicon-Based Sequencing of the CREBBP Gene as a Tool to Develop a Universal Platform-Independent Assay

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 se...

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Bibliographic Details
Published in:PloS one 2015-06, Vol.10 (6), p.e0129195-e0129195
Main Authors: Fuellgrabe, Marc W, Herrmann, Dietrich, Knecht, Henrik, Kuenzel, Sven, Kneba, Michael, Pott, Christiane, Brüggemann, Monika
Format: Article
Language:English
Subjects:
Accuracy
Computational Biology - methods
CREB-Binding Protein - genetics
Cyclic AMP response element-binding protein
Deoxyribonucleic acid
DNA
DNA sequencing
Exons
Exons - genetics
Gene sequencing
Genes
Genetic testing
Genome, Human - genetics
Genomes
Genomics
Genomics - methods
High-Throughput Screening Assays - methods
Humans
Mutation
Next-generation sequencing
Platforms
Protein binding
Workflow
Workflow software
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0129195