Characterisation of proviral integrations in corrected stem cells Visualisation of COL7A1 sequence by FISH analysis in corrected stem cells at passage.

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Characterisation of proviral integrations in corrected stem cells Visualisation of COL7A1 sequence by FISH analysis in corrected stem cells at passage XII (clone 6). Characterisation of proviral integrations in corrected stem cells Visualisation of COL7A1 sequence by FISH analysis in corrected stem cells at passage XII (clone 6). Five specific signals (red) were detected on five different chromosomes. As expected, a COL7A1 probe (red) hybridised to the two endogenous COL7A1 alleles located on the chromosomes 3 as identified by a specific centromeric probe (Cen 3) and to three other chromosomes identified as chromosomes 2, 11 and 22 by means of specific centromeric probes (Cen 2, Cen 11 and Cen 22, respectively). The latter corresponded to proviruses. Determination of proviral integration sites in corrected stem cell. Genomic DNA from clone 6 was submitted to whole‐genome sequencing together with PCR and subsequent Sanger sequencing to identify the integration sites to the base‐pair level. Mate pairs that span from the viral sequence to a human chromosome were extracted and used to estimate integration regions. The reads were mapped to the hg19 reference sequence. Three integration sites were uncovered: one in chromosome 2, one in chromosome 11 and one in chromosome 22, and targeted genes were identified. Analysis of the level of expression of targeted genes in corrected stem cells by quantitative RT–PCR. DARS was not significantly changed in clone 6 compared to the cells before transduction. Primers used annealed downstream of the proviral integration site. Error bars represent the standard deviation of three replicates. Identification of sequence abnormalities in the two alleles of the DARS‐targeted gene in clone 6 by NGS. Reads were mapped to the hg19 reference sequence. Small insertion–deletions and SNP calling were performed and compared to GWAS databases. The mapping highlighted two indels and one SNP in the DARS genomic sequence. The indels were not associated with disease and the SNP was synonymous (CCU–CCG both codons correspond to proline). Stéphanie Droz‐Georget Lathion et al. EMBO Mol Med. 2015;7:380-393 © as stated in the article, figure or figure legend