High-throughput sequencing of Epidermolysis Bullosa- and Epidermolytic Ichthyosis-associated genomic regions in immortalised keratinocytes (Ion Torrent amplicon panel)

Published: 28-02-2019| Version 2 | DOI: 10.17632/wntw75msnf.2
Contributor:
Oliver March

Description

We developed a comprehensive AmpliSeq panel for NGS analysis of EB and EI-asociated genomic loci. These included mutation hotspots and predicted off-target sites for gene editing agents. Some of the data generated was used to analyse the off-target and on-target capabilities of a KRT10-specific TALEN (6.21) in the development of a gene editing therapeutic option for Epidermolytic Ichthyosis. In this study, the KRT10 on-target 17q21.2 (KRT10 exon 6) and 8 in silico predicted off-targets 4q12 (non-coding), 6q21 (non-coding), 6p22.1 (non-coding), 4q31.3 (FHDC1 intron 6), 1p21 (non-coding), 2p25 (non-coding), 5q15 (non-coding), 1p36.3 (non-coding) were analysed. Other genomic loci were included on the amplicon panel. Although these were not analysed in this study, they are included in the provided metadata. NGS data was assessed in immortalised patient keratinocytes (EH31_Unmodified), TALEN-treated immortalised patient keratinocytes (EH31_T6.21) and a single cell clone derived from TALEN-treated immortalised patient keratinocytes (EH31_T6.21_AH2). Other samples were included on the amplicon panel, but are not in the provided metadata. Despite high on-target activity, TALEN 6.21 activity was not observed at off-target sites.

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Steps to reproduce

The panel and analysis was designed and performed as previously described (Kocher et al., 2017).