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

Area of Science:

• Ecology
• Genomics
• Molecular Biology

Background:

• RNA sequencing (RNA-Seq) is vital for gene expression analysis in wild populations.
• Sampling techniques and library preparation can influence RNA-Seq results.
• Understanding these technical variables is crucial for accurate gene expression studies in non-model organisms.

Purpose of the Study:

• To assess the impact of different fish sampling methods (dip netting vs. electrofishing) and tissue collection times on gene expression estimates.
• To compare the performance of two RNA-Seq library types: 3' RNA-Seq (QuantSeq) and whole mRNA-Seq (NEB).
• To determine the reliability of various field sampling strategies and RNA-Seq library types for gene expression studies.

Main Methods:

• Gene expression was analyzed in blood, gill, and muscle tissues of westslope cutthroat trout (Oncorhynchus clarkii lewisi).
• Two RNA-Seq library types, QuantSeq and NEB, were employed.
• Effects of dip netting versus electrofishing and immediate versus 5-minute post-euthanasia tissue harvesting were evaluated.

Main Results:

• No significant differences in gene expression were observed between sampling methods or tissue collection times for either library type.
• Whole mRNA-Seq (NEB) detected 31% more genes than 3' RNA-Seq (QuantSeq).
• 58% of detected genes showed significantly different expression levels between the NEB and QuantSeq library types.

Conclusions:

• Field sampling strategies for fish, including dip netting and electrofishing, do not significantly alter gene expression estimates.
• Whole mRNA-Seq (NEB) is more comprehensive than QuantSeq, offering advantages for detecting low-expressed genes, alternative splicing, and research in organisms with limited genomic resources.
• QuantSeq offers a cost-effective alternative, but NEB provides greater depth and breadth of gene detection.