Summary of gene expression analysis

A brief summary of our RNA-seq analysis.

RNA-seq

We used RNA sequencing (RNA-seq) - high-throughput sequence technology based on next-generation sequencing (NGS) - to analyse the transcriptome profiles affected by our experimental feed with different micronutrient compositions.

RNA-seq samples

We collected 17 male gonad samples at the final harvest stage for RNA-seq.

The number of samples for each group

  • L1 diet: 6 samples
  • L2 diet: 5 samples
  • L3 diet: 6 samples
No Name Diet Sex
1 G1 L2 M
2 G6 L3 M
3 G9 L3 M
4 G11 L1 M
5 G18 L2 M
6 G21 L1 M
7 G22 L1 M
8 G23 L1 M
9 G24 L1 M
10 G26 L3 M
11 G27 L3 M
12 G31 L2 M
13 G34 L2 M
14 G35 L2 M
15 G36 L3 M
16 G40 L3 M
17 G41 L1 M

Bioinformatics pipelne for RNA-seq

We used various bioinformatics algorithms and methods to analyse our RNA-seq samples. The following tools were those we used in our main RNA-seq pipeline.

Results

Overall diet effect

Clustering analysis showed no clear overall diet effects on the male gonad samples.

PCA bi-plot for RNA-seq samples
PCA (principal component analysis) bi-plot with top 500 high variance genes of RNA-seq counts with VST (variance stabilization transformation).

Differentially expressed genes

L3 diet affected gene expression profiles more than L2 diet in terms of the number of DEGs.

Identified DEGs for L2:L1 and L3:L1

  • L2 vs. L1: 6 DEGs
  • L3 vs. L1: 97 DEGs
Violin plot of L2:L1 DEGs Violin plot of L3:L1 DEGs
Volcano plots of differentially expressed genes in L2:L1 and L3:L1.

See What are DEGs? for more details about DEGs.

Significantly affected biological pathways

GSEA produces normalized enrichment scores (NESs) that indicate the trend of either up (positive value) or down (negative value) regulation of the identified pathways.

Enriched KEGG pathways with NESs by GSEA.
Pathway KEGG ID GSEA L2:L1 GSEA L3:L1
Ribosome biogenesis in eukaryotes sasa03008 - -2.68
RNA transport sasa03013 - -2.15
RNA polymerase sasa03020 - -2.48
DNA replication sasa03030 -2.31 -2.27
Spliceosome sasa03040 - -2.29
Nucleotide excision repair sasa03420 - -2.05
Mismatch repair sasa03430 -2.22 -2.20
Cytokine-cytokine receptor interaction sasa04060 2.14 2.19
Apoptosis sasa04210 2.00 -
ECM-receptor interaction sasa04512 2.22 -
Cell adhesion molecules sasa04514 2.18 -
Toll-like receptor signaling pathway sasa04620 2.16 -
NOD-like receptor signaling pathway sasa04621 2.14 -
C-type lectin receptor signaling pathway sasa04625 2.26 -
Intestinal immune network for IgA production sasa04672 - 2.13

Page links

Overview

DEG: differentially expressed gene

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