Rapid identification of heterozygous mutations in Drosophila melanogaster using genomic capture sequencing

Padlock capture was successfully used to identify novel mutations. (A) The genomic locus of twr and its alleles are shown. Mutations were detected within the Spase18-21 gene in three different twr alleles, twr¹, twr², and twr¹¹, using capture sequencing. Alleles twr¹ and twr¹¹ shared the same mutation of an A-to-T transition at position 3R:2,485,014. twr² was found to have a different transition of G to A at position 3R: 2,484,724. A P-element twr[05614] that failed to complement twr¹ was found by inverse PCR to be located 76 bp downstream from the Spase18-21 start site at position 3R:2,483,680. Compared with the wild type (B,D), twr¹/twr² transheterozygous adult flies (C,E) show rough eye phenotype and missing photoreceptors. (F) Alignment of reads at the position of the twr¹ mutation is shown. Alignment of capture sequencing reads covering the heterozygous mutation in the twr¹ is shown on the left. The mutated base pair is highlighted in red. Direct Sanger sequencing was performed to confirm the mutation in twr¹, with the heterozygous mutated base indicated by the arrow. The addition of 12 amino acids that were caused by the mutation are also shown. (G) Alignment of the SPASE18-21 amino acid sequence within several Drosophila species is shown, indicating a high degree of identity.