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 18 liver samples at the final harvest stage for RNA-seq.

The number of samples for each group

  • L1 diet: 6 samples
  • L2 diet: 6 samples
  • L3 diet: 6 samples
No Name Diet Sex
1 L1 L2 M
2 L6 L3 M
3 L9 L3 M
4 L11 L1 M
5 L18 L2 M
6 L21 L1 M
7 L22 L1 M
8 L23 L1 M
9 L24 L1 M
10 L26 L3 M
11 L27 L3 M
12 L31 L2 M
13 L32 L2 M
14 L34 L2 M
15 L35 L2 M
16 L36 L3 M
17 L40 L3 M
18 L41 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 clearly separated the liver samples into three groups by diet with L2 being in the middle.

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: 74 DEGs
  • L3 vs. L1: 245 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

Gene expression patterns in lipid metabolism were affected in a dose dependant manner (L3 < L2 < L1).

GSEA (gene set enrichment analysis) showed that most KEGG (Kyoto Encyclopedia of Genes and Genomes ) enriched pathways were down-regulated by micronutrient supplements.

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
Steroid biosynthesis sasa00100 -2.53 -2.76
Terpenoid backbone biosynthesis sasa00900 -2.31 -2.47
PPAR signalling pathway sasa03320 -1.58 -
Fatty acid metabolism sasa01212 -1.72 -2.2
Pyruvate metabolism sasa00620 - -1.86
Carbon metabolism sasa01200 -1.59 -1.97
Butanoate metabolism sasa00650 -1.94 -1.87
Glycine, serine and threonine metabolism sasa00260 -2.24 -1.71
Synthesis and degradation of ketone bodies sasa00072 -1.85 -1.75
Glutathione metabolism sasa00480 -1.61 -1.94

Page links

Overview

DEG: differentially expressed gene

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