Chiari Ylenia, Howard Leif, Moreno Nickolas, Relyea Scott, Dunnigan James, Boyer Matthew C, Kardos Marty, Glaberman Scott, Luikart Gordon
Department of Biology, George Mason University, Fairfax, Virginia, USA.
Flathead Lake Biological Station, Montana Conservation Genomics Laboratory, Division of Biological Science, University of Montana, Missoula, Montana, USA.
Mol Ecol Resour. 2023 May;23(4):803-817. doi: 10.1111/1755-0998.13757. Epub 2023 Feb 23.
RNA sequencing (RNA-Seq) is popular for measuring gene expression in non-model organisms, including wild populations. While RNA-Seq can detect gene expression variation among wild-caught individuals and yield important insights into biological function, sampling methods can also affect gene expression estimates. We examined the influence of multiple technical variables on estimated gene expression in a non-model fish, the westslope cutthroat trout (Oncorhynchus clarkii lewisi), using two RNA-Seq library types: 3' RNA-Seq (QuantSeq) and whole mRNA-Seq (NEB). We evaluated effects of dip netting versus electrofishing, and of harvesting tissue immediately versus 5 min after euthanasia on estimated gene expression in blood, gill, and muscle. We found no significant differences in gene expression between sampling methods or tissue collection times with either library type. When library types were compared using the same blood samples, 58% of genes detected by both NEB and QuantSeq showed significantly different expression between library types, and NEB detected 31% more genes than QuantSeq. Although the two library types recovered different numbers of genes and expression levels, results with NEB and QuantSeq were consistent in that neither library type showed differences in gene expression between sampling methods and tissue harvesting times. Our study suggests that researchers can safely rely on different fish sampling strategies in the field. In addition, while QuantSeq is more cost effective, NEB detects more expressed genes. Therefore, when it is crucial to detect as many genes as possible (especially low expressed genes), when alternative splicing is of interest, or when working with an organism lacking good genomic resources, whole mRNA-Seq is more powerful.
RNA测序（RNA-Seq）在测量非模式生物（包括野生种群）的基因表达方面很受欢迎。虽然RNA-Seq可以检测野生捕获个体之间的基因表达变异，并对生物学功能产生重要见解，但采样方法也会影响基因表达估计。我们使用两种RNA-Seq文库类型：3' RNA-Seq（QuantSeq）和全mRNA-Seq（NEB），研究了多个技术变量对非模式鱼类西坡割喉鳟（Oncorhynchus clarkii lewisi）估计基因表达的影响。我们评估了抄网捕捞与电捕，以及安乐死后立即采集组织与5分钟后采集组织对血液、鳃和肌肉中估计基因表达的影响。我们发现，两种文库类型的采样方法或组织采集时间之间，基因表达没有显著差异。当使用相同的血液样本比较文库类型时，NEB和QuantSeq都检测到的基因中有58%在文库类型之间表现出显著不同的表达，并且NEB比QuantSeq多检测到31%的基因。尽管两种文库类型回收的基因数量和表达水平不同，但NEB和QuantSeq的结果是一致的，即两种文库类型在采样方法和组织采集时间之间均未显示出基因表达差异。我们的研究表明，研究人员可以在野外安全地依赖不同的鱼类采样策略。此外，虽然QuantSeq更具成本效益，但NEB检测到更多表达的基因。因此，当尽可能多地检测基因（尤其是低表达基因）至关重要时，当对可变剪接感兴趣时，或者当研究缺乏良好基因组资源的生物体时，全mRNA-Seq更强大。
