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Array-based sequencing of filaggrin gene for comprehensive detection of disease-associated variants

doi: https://doi.org/10.1101/103416
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ABSTRACT

The filaggrin gene (FLG) is essential for skin differentiation and epidermal barrier formation with links to skin diseases, however it has a highly repetitive nucleotide sequence containing very limited stretches of unique nucleotides for precise mapping to reference genomes. Sequencing strategies using polymerase chain reaction (PCR) and conventional Sanger sequencing have been successful for complete FLG coding DNA sequence amplification to identify pathogenic mutations but this time-consuming, labour intensive method has restricted utility. Next-generation sequencing (NGS) offers obvious benefits to accelerate FLG analysis but standard re-sequencing techniques such as oligoprobe-based exome or customized targeted-capture can be expensive, especially for a single target gene of interest. We therefore designed a protocol to improve FLG sequencing throughput using a set of FLG-specific PCR primer assays compatible with microfluidic amplification, multiplexing and current NGS protocols. Using DNA reference samples with known FLG genotypes for benchmarking, this protocol is shown to be concordant for variant detection across different sequencing methodologies. We applied this methodology to analyze cohorts from ethnicities previously not studied for FLG variants and demonstrate usefulness for discovery projects. This comprehensive coverage sequencing protocol is labour-efficient and offers an affordable solution to scale up FLG sequencing for larger cohorts. Robust and rapid FLG sequencing can improve patient stratification for research projects and provide a framework for gene specific diagnosis in the future.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted January 26, 2017.
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Array-based sequencing of filaggrin gene for comprehensive detection of disease-associated variants
bioRxiv 103416; doi: https://doi.org/10.1101/103416
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Array-based sequencing of filaggrin gene for comprehensive detection of disease-associated variants
bioRxiv 103416; doi: https://doi.org/10.1101/103416

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