DOI: 10.5061/dryad.ns1rn8ptb
title: Influence of RNA-Seq library construction, sampling methods, and tissue harvesting time on gene expression estimation

description: 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.
description: Different tissues (gills, muscle and blood) were harvested from
 fry of westslope cutthroat trout immediately after death or after 5 min
 from death. Tissues were preserved in RNA later and extracted with QIazol
 (Qiagen). Transcriptome data were obtained following the 3' RNA
 Tag-Seq and whole mRNA-Seq library and sequencing procedures. Analyses of
 differential gene expression among sampling techniques and tissue
 harvesting time were carried out.
description: <strong>Table S1: Sample, RNA quality, gene counts, and
 library information. </strong>Sheet “<em>Samples
 All</em>” lists all samples collected (sample ID and Admera Health
 ID for QuantSeq and for NEB) with information about the treatment group
 they belong to, tissue type, sampling method, length and weight of the
 fish, RIN value, and RNA concentration. Sample size used for each
 comparison and divided for tissue type, treatment group, and library
 preparation is also indicated. Sheet “QuantSeq” lists all samples used for
 the QuantSeq library with the following information for each sample:
 treatment group, sample ID, Admera Health ID’s, tissue type, sampling
 method, RIN value, concentration, raw read count, read count after mapping
 the randomly selected 11 million reads, and percentage of uniquely mapped
 genes on the reference genome. Sheet “NEB” lists all samples used in the
 NEB library detailing for each sample the following: treatment group,
 sample ID, Admera Health ID’s (and new Admera Health ID if existing),
 tissue type, sampling method, RIN value, concentration, raw read count (PE
 and single), read count after mapping the randomly selected 40 million
 reads, and percentage of uniquely mapped genes on the reference
 genome. “<em>Null</em>" in some cells indicates empty
 cells -i.e., the samples were not processed due to a lower RIN than the
 cutoff and thus they do not have the Admera Health ID, or the data on fish
 weight and length are the same as in the line above since it is the same
 individual. "na" in some cells indicates that the RIN value
 could not be obtained. <strong>Table S2: Output
 results of the Differential Expression Analysis.</strong> Results of
 Differential Expression Analysis done with DESeq2 for all comparisons,
 each of them presented on a separate sheet. Groups 1, 2, and 3 refer to
 sampling by netting, electrofishing, and electrofishing with processing 5
 min after euthanasia, respectively. <strong>Table
 S3: Summary of gene expression patterns for different sampling methods and
 tissue types.</strong> The total numbers of genes with detectable
 expression for each sampling/tissue comparison are indicated along with
 the number and proportion of genes with significantly higher gene
 expression in one of the two tissues being compared for each sampling
 method.