Characterisation of a LINE-1 Insertion in the RP1 Gene by Targeted Adaptive Nanopore Sequencing in a Family with Retinitis Pigmentosa

Figure 4

Breakpoint PCR and the Sanger sequencing confirm the L1 insertion. (a) A schematic figure of the L1 insertion (green) in exon 4 of the RP1 gene (NM_006269.2) (purple). The pA-tail of L1 is shown in orange, and the target site duplication (TSD) is in blue. Primers were designed to amplify the 5 and 3 breakpoints (BP), and the PCR amplicons over the 5 BP and 3 BP are indicated, as well as the sequencing directions used for the Sanger sequencing. (b) PCR reactions representing the 5 BP and 3 BP of the L1 insertion. Specific PCR products of the expected size are indicated (arrowhead). Unspecific PCR products likely caused by L1-specific primers are indicated (asterisk). Lanes: M: marker; UC: unrelated healthy control; II.3: affected relative; II.4: unaffected relative; III.1 and III.2: patients; N: negative PCR control. Products of the expected size were amplified from the DNA samples of the affected patients (III.1, III.2) and the affected relative (II.3). No specific PCR product was amplified from the unrelated control or the unaffected relative (II.4), indicating that they lacked the L1 insertion. (c) Representative schematic figures of the insertion of BP Sanger’s sequencing results. Sequencing over the 5 BP (top panel) was done by directly sequencing the PCR product isolated from agarose gel. Because of the 3 terminal poly-A (pA) tract of L1, the PCR product was cloned into a plasmid vector before sequencing (middle and bottom panels). The sequence preceding the pA tract aligned with the L1 subfamily L1M3_orf2 subfamily in antisense orientation and the sequence succeeding the pA tract aligned with RP1, including the TSD. For coding of background colouring of the sequences, see (a). The premature stop codon introduced by the L1 insertion is underlined (top panel).

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