Amplicon genome fishing (AGF): a rapid and efficient method for sequencing target cis-regulatory regions in nonmodel organisms.

Cis-regulatory sequences play a crucial role in regulating gene expression and are evolutionary hot spots that drive phenotypic divergence among organisms. Sequencing some cis-regulatory regions of interest in many different species is common in comparative genetic studies. For nonmodel organisms lacking genomic data, genome walking is often the preferred method for this type of application. However, applying genome walking will be laborious and time-consuming when the number of cis-regulatory regions and species to be analyzed is large. In this study, we propose a novel method called amplicon genome fishing (AGF), which can isolate and sequence cis-regulatory regions of interest for any organism. The main idea of the AGF method is to use fragments amplified from the target cis-regulatory regions as enrichment baits to capture and sequence the whole target cis-regulatory regions from genomic library pools. Unlike genome walking, the AGF method is based on hybridization capture and high-throughput sequencing, which makes this method rapid and efficient for projects where some cis-regulatory regions have to be sequenced for many species. We used human amplicons as capture baits and successfully sequenced five target enhancer regions of Homo sapiens, Mus musculus, Gallus gallus, and Xenopus tropicalis, proving the feasibility and repeatability of AGF. To show the utility of the AGF method in real studies, we used it to sequence the ZRS enhancer, a cis-regulatory region associated with the limb loss of snakes, for twenty-three vertebrate species (includes many limbless species never sequenced before). The newly obtained ZRS sequences provide new perspectives into the relationship between the ZRS enhancer's evolution and limb loss in major tetrapod lineages. [ABSTRACT FROM AUTHOR]