Department of Genetics, Microbiology and Statistics and IRBio, University of Barcelona, Barcelona, Spain.
Center for Advanced Studies of Blanes (CEAB-CSIC), Blanes, Girona, Spain.
Mol Ecol Resour. 2020 May;20(3). doi: 10.1111/1755-0998.13144. Epub 2020 Mar 4.
High-throughput sequencing has revolutionized population and conservation genetics. RAD sequencing methods, such as 2b-RAD, can be used on species lacking a reference genome. However, transferring protocols across taxa can potentially lead to poor results. We tested two different IIB enzymes (AlfI and CspCI) on two species with different genome sizes (the loggerhead turtle Caretta caretta and the sharpsnout seabream Diplodus puntazzo) to build a set of guidelines to improve 2b-RAD protocols on non-model organisms while optimising costs. Good results were obtained even with degraded samples, showing the value of 2b-RAD in studies with poor DNA quality. However, library quality was found to be a critical parameter on the number of reads and loci obtained for genotyping. Resampling analyses with different number of reads per individual showed a trade-off between number of loci and number of reads per sample. The resulting accumulation curves can be used as a tool to calculate the number of sequences per individual needed to reach a mean depth ≥20 reads to acquire good genotyping results. Finally, we demonstrated that selective-base ligation does not affect genomic differentiation between individuals, indicating that this technique can be used in species with large genome sizes to adjust the number of loci to the study scope, to reduce sequencing costs and to maintain suitable sequencing depth for a reliable genotyping without compromising the results. Here, we provide a set of guidelines to improve 2b-RAD protocols on non-model organisms with different genome sizes, helping decision-making for a reliable and cost-effective genotyping.
高通量测序技术彻底改变了群体遗传学和保护遗传学。RAD 测序方法，如 2b-RAD，可用于缺乏参考基因组的物种。然而，跨类群转移协议可能会导致结果不佳。我们在两种具有不同基因组大小的物种（红海龟 Caretta caretta 和尖嘴鲈 Diplodus puntazzo）上测试了两种不同的 IIB 酶（AlfI 和 CspCI），以制定一套指导方针，在非模式生物中改进 2b-RAD 协议，同时优化成本。即使使用降解样本，也能获得良好的结果，这表明 2b-RAD 在 DNA 质量较差的研究中具有价值。然而，文库质量被发现是获得基因型所需的读取数量和基因座数量的关键参数。不同个体读取数的重采样分析显示出在基因座数量和每个样本的读取数之间的权衡。所得的积累曲线可作为一种工具，用于计算每个个体所需的序列数，以达到 20 个读取以上的平均深度，从而获得良好的基因型结果。最后，我们证明了选择性碱基连接不会影响个体之间的基因组分化，这表明该技术可用于基因组较大的物种，以根据研究范围调整基因座数量，降低测序成本，并保持适合的测序深度，以进行可靠的基因型分析，而不会影响结果。在这里，我们提供了一套针对不同基因组大小的非模式生物的 2b-RAD 协议改进指南，帮助做出可靠和具有成本效益的基因型分析决策。
