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Influence of RNA-Seq library construction, sampling methods, and tissue harvesting time on gene expression estimation
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  • Ylenia Chiari,
  • Leif Howard,
  • Nickolas Moreno,
  • Scott Relyea,
  • James Dunnigan,
  • Matthew Boyer,
  • Marty Kardos,
  • Scott Glaberman,
  • Gordon Luikart
Ylenia Chiari
George Mason University

Corresponding Author:ychiari@gmu.edu

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Leif Howard
University of Montana Missoula Division of Biological Sciences
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Nickolas Moreno
George Mason University
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Scott Relyea
Montana Fish Wildlife and Parks
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James Dunnigan
Montana Fish Wildlife and Parks
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Matthew Boyer
University of Montana
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Marty Kardos
National Oceanic and Atmospheric Center
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Scott Glaberman
George Mason University
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Gordon Luikart
University of Montana Missoula Division of Biological Sciences
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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.
01 May 2022Submitted to Molecular Ecology Resources
22 May 2022Reviewer(s) Assigned
17 Jun 2022Review(s) Completed, Editorial Evaluation Pending
25 Jul 2022Editorial Decision: Revise Minor
15 Dec 20221st Revision Received
15 Dec 2022Review(s) Completed, Editorial Evaluation Pending
17 Jan 2023Editorial Decision: Accept