Influence of RNA-Seq library construction, sampling methods, and tissue
harvesting time on gene expression estimation
Abstract
RNA sequencing (RNA-Seq) is a popular method for measuring gene
expression in non-model organisms, including wild populations. While
RNA-Seq can measure gene expression variation among wild-caught
individuals and can yield important biological insights into organism
function, sampling methods may also 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 and whole mRNA-Seq. 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 detected 30% more genes with whole mRNA-Seq than with 3’
RNA-Seq and found that 58% of genes were significantly differently
expressed between 3’ RNA-Seq and whole mRNA-Seq. Our findings indicate
that 3’ RNA-Seq and whole mRNA-Seq are robust to the technical variables
related to the field sampling approaches tested here with a lack of
differential gene expression among sampling methods and tissue
collection time after euthanasia. However, we found that gene expression
varied based on which RNA-Seq library type was used on the same set of
samples. Our study suggests researchers could safely rely on different
fish sampling strategies in the field and save money and analyze more
individuals using 3’ RNA-Seq, but should use whole mRNA-Seq when working
with a species without good genomic resources, and when maximizing the
number of genes identified and detecting alternative splicing are
important.