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Gene expression estimates: Influence of sequencing library construction, fish sampling methods, and tissue harvesting time
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  • Nickolas Moreno,
  • Leif Howard,
  • Scott Relyea,
  • James Dunnigan,
  • Matthew Boyer,
  • Marty Kardos,
  • Scott Glaberman,
  • Gordon Luikart,
  • Ylenia Chiari
Nickolas Moreno
George Mason University
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Leif Howard
University of Montana Missoula Division of Biological Sciences
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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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Ylenia Chiari
George Mason University
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Abstract

RNA sequencing (RNA-Seq) is becoming a popular method for measuring gene expression in non-model organisms, including wild populations sampled in the field. While RNA-Seq can be used to measure gene expression variation among wild-caught individuals and can yield important biological insights into organismal function, technical variables may also influence gene expression estimates. We examined the influence of multiple technical variables on estimated gene expression in a non-model fish species, the westslope cutthroat trout (Oncorhynchus clarkii lewisi), using two RNA-Seq methods: 3’ RNA-Seq and whole mRNA-Seq. We evaluated the effects of dip netting versus electrofishing, and of harvesting tissue immediately versus 5 minutes after euthanasia on estimated gene expression in blood, gill, muscle, and liver. We found higher RNA degradation in the liver compared to the other tissues. There were fewer expressed genes in blood compared to gill and muscle. We found no difference in gene expression among sampling methods or due to a delay in tissue collection. However, we detected fewer genes with 3’ RNA-Seq than with whole mRNA-Seq and found statistically significant differences in gene expression between 3’ RNA-Seq and whole mRNA-Seq. The magnitude and direction of these differences does not appear to be dependent on gene type or length. Our findings indicate that RNA-Seq is robust to the technical variables related to the field sampling techniques tested here but varies based on the tissue sampled and the RNA-Seq library used. This study advances understanding of usefulness of RNA-Seq to study gene expression variation in evolution, ecology, and conservation.