当前位置: X-MOL 学术Mol. Ecol. Resour. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Helping decision making for reliable and cost-effective 2b-RAD sequencing and genotyping analyses in non-model species.
Molecular Ecology Resources ( IF 7.7 ) Pub Date : 2020-03-04 , DOI: 10.1111/1755-0998.13144
Anna Barbanti 1 , Hector Torrado 1, 2 , Enrique Macpherson 2 , Luca Bargelloni 3 , Rafaella Franch 3 , Carlos Carreras 1 , Marta Pascual 1
Affiliation  

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.

中文翻译:

帮助决策者在非模型物种中进行可靠且经济高效的2b-RAD测序和基因分型分析。

高通量测序彻底改变了种群和保护遗传学。RAD测序方法(例如2b-RAD)可用于缺少参考基因组的物种。但是,跨类群传输协议可能会导致较差的结果。我们在两个具有不同基因组大小的物种(海龟和尖嘴鲷鲷鱼)上测试了两种不同的IIB酶(AlfI和CspCI),以建立一套指导方针,以改进针对非模式生物的2b-RAD方案,同时优化费用。即使使用降解的样品也获得了良好的结果,显示了2b-RAD在DNA质量较差的研究中的价值。但是,发现文库质量是获得基因分型的读数和基因座数量的关键参数。对每个个体使用不同数量的读数进行的重采样分析显示,在基因座数量与每个样品的读数数量之间进行权衡。所得的积累曲线可以用作计算平均深度≥20读数以获取良好的基因分型结果所需的每个个体的序列数的工具。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具有大基因组大小的物种中,以将基因座的数量调整至研究范围,降低测序成本并维持合适的测序在不影响结果的前提下进行可靠的基因分型。在这里,我们提供了一套指南,以改进针对具有不同基因组大小的非模型生物的2b-RAD方案,
更新日期:2020-03-04
down
wechat
bug