Summary of DNA methylation analysis

A brief summary of our DNA methylation analysis.

RRBS

We used RRBS (reduced representation bisulfite sequencing) to analyse the DNA methylation profiles affected by our experimental feed with different micronutrient compositions. RRBS utilises restriction enzymes to target CpG rich regions in the genome. We used two different enzymes for our RRBS analysis.

Restriction enzymes used for RRBS

  1. MspI (recognition sequence : 5' CCGG)
  2. TaqI (recognition sequence : 5' TCGA)

RRBS samples

We collected 9 male gonad samples at the final harvest stage for RRBS sequencing.

The number of samples for each group

  • L1 diet: 3 samples
  • L2 diet: 3 samples
  • L3 diet: 3 samples
No Name Diet Sex
G11 L1 M
G24 L1 M
G41 L1 M
G1 L2 M
G18 L2 M
G31 L2 M
G26 L3 M
G27 L3 M
G40 L3 M

Definition of genomic regions

Functions of DNA methylation can be different depending on the types of regions where methylation occurs. We split the genome into seven different regions for our RRBS analysis.

Genomic regions for RRBS read mapping

  1. Exon
  2. Intron
  3. P250 (proximal promoter)
  4. P1K (promoter)
  5. P5K (distal promoter)
  6. Flanks (potential enhancer region)
  7. IGR (intergenic region)
Genomic regions for RRBS read alignment
Definition of genomic regions for RRBS read alignment.

Bioinformatics pipelne for RRBS

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

Results

Overall diet effect

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

PCA bi-blot for RRBS samples
PCA (principal component analysis) bi-plot for clustering the 9 RRBS samples.

Differentially methylated CpG sites

There were no noticeable differences between L2:L1 and L3:L1 as well as hypo- and hyper-methylation in terms of the number of DMCs.

Identified DMCs for L2:L1 and L3:L1

  • L2 vs. L1: 27433 DMCs
  • L3 vs. L1: 26995 DMCs
Violin plots of DMCs
Violin plots of DMCs.

See What are DMCs? for more details about DMCs.

Significantly affected biological pathways

ORA (over representation analysis) on KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways showed that micronutrient supplement significantly affected DNA methylation profiles in various pathways mainly through DNA methylation in their gene bodies.

Enriched KEGG pathways by ORA (over representation analysis).
Pathway KEGG ID L2L1 L3L1
Purine metabolism sasa00230 - Gene body (65/1898)
Lysine degradation sasa00310 - Exon (15/545)
Mucin type O-glycan biosynthesis sasa00512 P+GB (25/2311) -
Various types of N-glycan biosynthesis sasa00513 Gene body (25/1958), Intron (23/1516) Intron (24/1490)
Other types of O-glycan biosynthesis sasa00514 RS+GB (38/2818), P+GB (32/2311), Gene body (27/1958) -
Glycosaminoglycan biosynthesis - heparan sulfate / heparin sasa00534 RS+GB (29/2818), P+GB (28/2311), Gene body (26/1958), Intron (24/1516) RS+GB (31/2725), P+GB (27/2219), Gene body (25/1898), Intron (22/1490)
Glycerolipid metabolism sasa00561 RS+GB (43/2818), P+GB (37/2311), Gene body (31/1958) -
Inositol phosphate metabolism sasa00562 RS+GB (55/2818), Gene body (42/1958) -
ErbB signaling pathway sasa04012 RS+GB (83/2818), P+GB (73/2311), Gene body (66/1958), Intron (59/1516) RS+GB (78/2725), P+GB (65/2219), Gene body (61/1898), Intron (54/1490)
Phosphatidylinositol signaling system sasa04070 RS+GB (70/2818), P+GB (62/2311), Gene body (58/1958), Intron (47/1516) -
Cellular senescence sasa04218 P5K (25/357) -
Adrenergic signaling in cardiomyocytes sasa04261 RS+GB (160/2818), P+GB (139/2311), Gene body (122/1958), Intron (104/1516) RS+GB (133/2725), P+GB (118/2219), Gene body (103/1898), Intron (83/1490)
Vascular smooth muscle contraction sasa04270 RS+GB (89/2818), P+GB (76/2311), Gene body (66/1958) -
Wnt signaling pathway sasa04310 RS+GB (129/2818), P+GB (112/2311), Gene body (98/1958) RS+GB (125/2725), P+GB (106/2219), Gene body (91/1898)
Apelin signaling pathway sasa04371 RS+GB (102/2818), P+GB (91/2311), Gene body (77/1958), Intron (64/1516) -
ECM-receptor interaction sasa04512 RS+GB (78/2818), P+GB (70/2311), Gene body (63/1958), Exon (23/570), Intron (50/1516) RS+GB (66/2725), P+GB (61/2219), Gene body (58/1898), Exon (23/545), Intron (42/1490)
Cell adhesion molecules sasa04514 P+GB (98/2311), Gene body (86/1958), Intron (72/1516) P+GB (96/2219), Gene body (87/1898), Intron (73/1490)
Adherens junction sasa04520 RS+GB (87/2818), P+GB (77/2311), Gene body (72/1958), Exon (25/570), Intron (55/1516) RS+GB (78/2725), P+GB (68/2219), Gene body (60/1898), Intron (49/1490)
Gap junction sasa04540 RS+GB (73/2818), P+GB (64/2311), Gene body (61/1958), Intron (45/1516) -
Insulin signaling pathway sasa04910 P+GB (80/2311) -
GnRH signaling pathway sasa04912 RS+GB (83/2818), P+GB (76/2311), Gene body (69/1958), Intron (57/1516) Intron (47/1490)
Melanogenesis sasa04916 RS+GB (90/2818), P+GB (80/2311), Gene body (72/1958), Intron (53/1516) -
Adipocytokine signaling pathway sasa04920 - P+GB (49/2219), Intron (34/1490)

Page links

Overview

DMC: differentially methylated CpG site

DMG: differentially methylated gene

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