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

Abstract

One of the key advantages of using Drosophila melanogaster as a genetic model organism is the ability to conduct saturation mutagenesis screens to identify genes and pathways underlying a given phenotype. Despite the large number of genetic tools developed to facilitate downstream cloning of mutations obtained from such screens, the current procedure remains labor intensive, time consuming, and costly. To address this issue, we designed an efficient strategy for rapid identification of heterozygous mutations in the fly genome by combining rough genetic mapping, targeted DNA capture, and second generation sequencing technology. We first tested this method on heterozygous flies carrying either a previously characterized dac(5) or sens(E2) mutation. Targeted amplification of genomic regions near these two loci was used to enrich DNA for sequencing, and both point mutations were successfully identified. When this method was applied to uncharacterized twr mutant flies, the underlying mutation was identified as a single-base mutation in the gene Spase18-21. This targeted-genome-sequencing method reduces time and effort required for mutation cloning by up to 80% compared with the current approach and lowers the cost to <$1000 for each mutant. Introduction of this and other sequencing-based methods for mutation cloning will enable broader usage of forward genetics screens and have significant impacts in the field of model organisms such as Drosophila.

Figure 1.

Two known mutations were detected using padlock capture sequencing. (A) Flowchart of mutation detection by padlock capture and next-generation sequencing technologies. Padlock capture technology requires probes that contain two target-specific capturing arms (green) connected by a common linker (red). The unique targeting arms of individual targeting oligonucleotides are designed to hybridize immediately upstream and downstream from each exon (purple bars) of interest. Hybridization to genomic DNA is followed by an enzymatic gap filling and ligation step, such that a copy of the sequence of interest is incorporated into a circle (purple dashed line). Then the enriched DNA is PCR amplified and is used to prepare libraries for the next-generation platform sequencing. (B) sens^E2 and (C) dac⁵ mutation detection. Reads alignment at the mutation is shown between the vertical lines. Each read is drawn as a gray line, and bases that are different from the reference are colored. Sanger sequencing is conducted to confirm the mutation with the heterozygous mutated base indicated by the arrow.

Figure 2.

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.