Ramos-Onsins Sebastian E, Guirao-Rico Sara, Hafez Ahmed, Ferretti Luca
Plant and Animal Genomics Program, Centre for Research in Agricultural Genomics (CRAG), Consejo Superior de Investigaciones Científicas-Institute of Agrifood Research and Technology-Autonomous University of Barcelona-University of Barcelona, Bellaterra, Spain.
Departament de Genètica, Microbiologia i Estadística, Universitat de Barcelona, Barcelona, Spain.
Methods Mol Biol. 2025;2935:51-66. doi: 10.1007/978-1-0716-4583-3_3.
Pool sequencing has emerged as a valuable approach in ecological studies, particularly when dealing with very small organisms (with limited amount of DNA available), when distinguishing individual organisms is a challenge (e.g., in colonies, microbiome), when there is a trade-off between the sequencing cost and the number of individuals to sequence, when the main goal is to estimate nucleotide variability and variant frequency patterns at the population level (that is, when individual information is not required). Estimates of variability can be efficiently explored by analyzing sequences of pooled individuals sampled from the population. When using this approach, the number of pooled individuals and the mean read depth are key choices in the experimental design.The software npstat calculates different estimates of nucleotide variability and neutrality tests.It also calculates the number of synonymous and nonsynonymous variants and the proportion of beneficial substitutions (alpha) using the MKT approach when GTF annotation file and an outgroup is provided.
混合测序已成为生态研究中的一种有价值的方法,特别是在处理非常小的生物(可用DNA量有限)时,在区分个体生物具有挑战性时(例如在菌落、微生物组中),在测序成本与要测序的个体数量之间存在权衡时,当主要目标是估计群体水平上的核苷酸变异性和变异频率模式时(即当不需要个体信息时)。通过分析从群体中采样的混合个体的序列,可以有效地探索变异性估计。使用这种方法时,混合个体的数量和平均读长深度是实验设计中的关键选择。软件npstat计算核苷酸变异性的不同估计值和中性检验。当提供GTF注释文件和一个外群时,它还使用MKT方法计算同义变体和非同义变体的数量以及有益替换的比例(α)。