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.

顺式调节序列在调节基因表达中起关键作用，并且是驱动生物之间表型差异的进化热点。在比较遗传学研究中，常见的是在许多不同物种中对某些顺式调控区域进行测序。对于缺乏基因组数据的非模型生物，基因组遍历通常是此类应用程序的首选方法。但是，当要分析的顺式调控区和物种数量很多时，应用基因组步移将是费力且费时的。在这项研究中，我们提出了一种称为扩增子基因组捕鱼（AGF）的新方法，该方法可以分离和测序顺式-任何生物体感兴趣的调控区域。AGF方法的主要思想是使用从目标顺式调控区扩增的片段作为富集诱饵，从基因组文库中捕获并测序整个目标顺式调控区。与基因组漫游不同，AGF方法基于杂交捕获和高通量测序，这使得该方法对于需要对许多物种进行某些顺式调控区域进行测序的项目而言，是快速有效的。我们使用人类扩增子作为捕获诱饵，并成功地测序了智人，小家鼠，鸡胆和热带爪蟾的5个目标增强子区。，证明了AGF的可行性和可重复性。为了显示AGF方法在实际研究中的实用性，我们使用它对23种脊椎动物（包括许多以前从未测序过的无肢物种）进行ZRS增强子（与蛇的肢体丢失相关的顺式调控区域）的测序。新获得的ZRS序列为ZRS增强子的进化与主要四足动物谱系的肢体丢失之间的关系提供了新的视角。