3.1 RNA and raw sequencing data quality statistics
Out of the 120 samples for which RNA was extracted, 86 had a RIN value (a measure of RNA integrity) equal or above 8.8. Little variation in RIN scores was observed among the sampled tissues and sampling methods (Table 1 and data on Dryad). Mean and standard deviation for RIN values for the three tissues were: 9.6±0.22 (blood), 9.2±0.40 (muscle), 9.0±1 (gill). Mean and standard deviation for RIN values for the three treatment groups: 9.2±0.43 (dip netting), 9.3±0.34 (electrofishing), and 9.2±1.06 (tissue harvesting after 5 minutes). We found no differences in RIN values among groups (F = 0.299, df = 2, p = 0.74) and in RIN values among tissues within each group (F = 0.595, df = 4,p = 0.67).
Table S1 on Dryad ). The final number of reads per individual for QuantSeq libraries ranged from 11 million to 15.6 million (mean = 12.88 million ± 0.67; data on Dryad). On average, of the 11 million reads randomly selected for each sample, we obtained around 77% of uniquely mapped reads on the rainbow trout (O. mykiss) genome independently of the sampling method used (range: 67.7 ‐ 86.3%, Table S1 on Dryad .
RNA sequencing from the 14 NEB samples (blood only) yielded a total of 564 million reads for individuals captured by net (mean = 112.9 million+ 13.95; N = 5), 563.4 million reads for samples collected by electrofishing and sampled immediately (mean = 112.7 million +22.4; N = 5), and 350.4 million reads from electrofishing samples processed after 5 minutes (mean = 87.6 million + 7.4; N = 4). The final number of reads per individual ranged from 77.8 to 148.8 million reads (mean = 105.6 million ± 19.1). On average, of the 40 million reads randomly selected for each sample, we obtained on average 75% of uniquely mapped reads on the O. mykiss genome (Table S1 on Dryad).
Mapping reads on the rainbow trout (O. mykiss) genome and looking at the output of DESeq2 between the two library types and for the same samples, we found that NEB detected 30% more genes than QuantSeq (Table S2 on Dryad). A gene was considered to be detected/expressed when basemean was different from 0. Specifically, for the 14 blood samples for which the two different types of RNA-Seq libraries were built, we found that NEB and QuantSeq detected approximately 35K and 26K genes, respectively, which mapped onto the annotated rainbow trout (O. mykiss ) genome. 25K genes were detected by both library types. However, 9K genes were detected by NEB but not QuantSeq, and 1K genes were detected by QuantSeq but not NEB. Presence/absence of genes detected by one or the other library type is independent of gene transcript length (Figure 1).