Influence of RNA-Seq library construction, sampling methods, and tissue harvesting time on gene expression estimation

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 may
influence 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 minutes 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 QuantSeq and NEB recovered
different numbers of genes and expression levels, there were no
differences in gene expression between sampling methods and tissue
harvesting time for either library type. 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.