Sequence-based detection and breakpoint assembly of polymorphic inversions

Abstract

Inversion polymorphisms have occupied a privileged place in Drosophila genetic research since their discovery in the 1920s. Indeed, inversions seem to be nearly ubiquitous, and the majority of species that have been thoroughly surveyed have been found to be polymorphic for one or more chromosomal inversions. Despite enduring interest, however, inversions remain difficult to study because their effects are often cryptic, and few efficient assays have been developed. Even in Drosophila melanogaster, in which inversions can be reliably detected and have received considerable attention, the breakpoints of only three inversions have been characterized molecularly. Hence, inversion detection and assay design remain important unsolved problems. Here, we present a method for identification and local de novo assembly of inversion breakpoints using next-generation paired-end reads derived from D. melanogaster isofemale lines. PCR and cytological confirmations demonstrate that our method can reliably assemble inversion breakpoints, providing tools for future research on D. melanogaster inversions as well as a framework for detection and assay design of inversions and other chromosome aberrations in diverse taxa.

Figure 1

Mapping positions of reads used for de novo assembly of the breakpoint sequences. (Top) Reads mapping positions on the reference sequence. (Bottom) Their inferred positions on the inverted haplotype. Reads pairs that span the breakpoint are shown in shades of blue, while reads for which one end maps and the pair crosses the junction on the reference sequence are shown in shades of green. All reads shown are used to produce de novo assemblies. For simplicity, reads corresponding to this inversion’s other breakpoint are not shown.

Figure 2

We found two types of inversion breakpoints: cut-and-paste breakpoints (top), and staggered breakpoints (bottom), which create inverted duplications at the breakpoints. The duplicated regions are shown as purple and blue “genes.” PCR primers for the standard (in orange) and inverted (in teal) arrangements were designed on the basis of assembled contigs that span the breakpoints and amplify a unique product for either arrangement.