3.1 Quality control of sequencing data
The sequencing data from this research underwent rigorous quality control. All samples showcased a Total Mapped rate of over 84.28%, with the proportion of clean reads exceeding 94.19% (details presented in Table 1). This attests to the high caliber of our sequencing data, meeting the prerequisites for subsequent analyses.
Table 1
Quality control of sequencing data
Sample
|
Number of
Clean Reads
|
Clean Bases
|
Clean Reads
(%)
|
Total Mapped
|
CG1_1
|
41878340
|
6281751000
|
94.52
|
38487510 (91.90%)
|
CG1_2
|
41631208
|
6244681200
|
94.19
|
38484793 (92.44%)
|
PL1_1
|
39747534
|
5962130100
|
94.46
|
36406707 (91.59%)
|
PL1_2
|
40388704
|
6058305600
|
94.3
|
36995841 (91.60%)
|
PLM1_1
|
38129124
|
5719368600
|
94.39
|
34439028 (90.32%)
|
PLM1_2
|
41903456
|
6285518400
|
94.54
|
38074131 (90.86%)
|
WT1_1
|
40630984
|
6094647600
|
94.49
|
37250401 (91.68%)
|
WT1_2
|
40604698
|
6090704700
|
94.43
|
37332550 (91.94%)
|
WTM1_1
|
35421904
|
5313285600
|
94.75
|
32599990 (92.03%)
|
WTM1_2
|
35646052
|
5346907800
|
94.62
|
32934318 (92.39%)
|
CG2_1
|
49688468
|
7453270200
|
94.77
|
43222933 (86.99%)
|
CG2_2
|
36186062
|
5427909300
|
94.34
|
31207951 (86.24%)
|
PL2_1
|
41544190
|
6231628500
|
94.66
|
35549612 (85.57%)
|
PL2_2
|
39395136
|
5909270400
|
94.29
|
33204098 (84.28%)
|
PLM2_1
|
39654000
|
5948100000
|
94.66
|
34018607 (85.79%)
|
PLM2_2
|
35676028
|
5351404200
|
94.42
|
31166837 (87.36%)
|
WT2_1
|
41107440
|
6166116000
|
94.3
|
35902911 (87.34%)
|
WT2_2
|
42055822
|
6308373300
|
94.32
|
36980670 (87.93%)
|
WTM2_1
|
37243672
|
5586550800
|
94.77
|
32843359 (88.19%)
|
WTM2_2
|
35473958
|
5321093700
|
94.7
|
31286636 (88.20%)
|
CG1-1, CG12: Control group liver samples.
PL1_1, PL1_2: Liver samples from peak oocyst output (120h) post-inoculation with the PL of E.media.
PLM1_1, PLM1_2: Liver samples from the end period of oocyst output (168h) after inoculation with the PL of E.media.
WT1_1, WT1_2: Liver samples from peak oocyst output (192h) post-inoculation with the WT of E.media.
WTM1_1, WTM1_2: Liver samples from the end period of oocyst output (216h) post-inoculation with the WT of E.media.
CG2_1, CG2_2: Control group duodenal samples.
PL2_1, PL2_2: Duodenal samples from peak oocyst output (120h) post-inoculation with the PL of E.media.
PLM2_1, PLM2_2: Duodenal samples from the end period of oocyst output (168h) after inoculation with the PL of E.media.
WT2_1, WT2_2: Duodenal samples from peak oocyst output (192h) post-inoculation with the WT of E.media.
WTM2_1, WTM2_2: Duodenal samples from the end period of oocyst output (216h) post-inoculation with the WT of E.media.
3.4 Biological Function Analysis of Differentially Expressed Genes
Based on the GO database, the differentially expressed genes were functionally categorized. The top 20 GO entries under each functional category are shown in the figure below. In liver samples: For PL1 vs CG1, entries that are significantly enriched (P < 0.05) include immune system process, regulation of immune system process, immune response, defense response, cellular response to chemical stimulus, response to external stimulus, lipid metabolic process, small molecule metabolic process, cell activation, and so on(Fig. 3A). For PLM1 vs CG1, entries that are significantly enriched (P < 0.05) include binding, cellular response to chemical stimulus, oxidation-reduction process, response to chemical, small molecule metabolic process, cellular response to an organic substance, and cytoplasmic part, among others(Fig. 3B). For WT1 vs CG1, entries that are significantly enriched (P < 0.05) include small molecule metabolic process, oxoacid metabolic process, carboxylic acid metabolic process, organic acid metabolic process, carboxylic acid catabolic process, alpha-amino acid metabolic process, lipid metabolic process, arginine metabolic process, cellular response to oxygen-containing compound, etc (Fig. 3C). For WTM1 vs. CG1, entries that are significantly enriched (P < 0.05) encompass integrin binding, extracellular region, negative regulation of plasminogen activation, blood vessel development, cell adhesion molecule binding, negative regulation of lipoprotein lipase activity, insulin-like growth factor binding, vasculature development, regulation of endothelial cell apoptotic process, metallocarboxypeptidase activity, and carboxypeptidase activity, etc(Fig. 3D).
In the duodenal samples: For PL2 vs CG2, entries that are significantly enriched (P < 0.05) include catalytic activity, oxidation-reduction process, small molecule metabolic process, organic acid metabolic process, oxoacid metabolic process, cofactor binding, small molecule biosynthetic process, monocarboxylic acid metabolic process, ion binding, lipid metabolic process, and apical part of the cell, etc(Fig. 3E). For PLM2 vs CG2, entries that are significantly enriched (P < 0.05) are immune system process, regulation of immune system process, leukocyte activation, cell activation, lymphocyte activation, positive regulation of immune system process, T cell activation, regulation of immune response, immune effector process, defense response, and lymphocyte-mediated immunity, etc(Fig. 3F). For WT2 vs. CG2, significantly enriched entries (P < 0.05) encompass the extracellular region part, extracellular region, small molecule metabolic process, organic acid metabolic process, oxoacid metabolic process, oxidoreductase activity, carboxylic acid metabolic process, extracellular space, catalytic activity, lipid metabolic process, and extracellular matrix, etc(Fig. 3G). For WTM2 vs. CG2, entries that are significantly enriched (P < 0.05) include the oxidation-reduction process, anion transport, lipid metabolic process, lipid localization, catalytic activity, oxidoreductase activity, ion transport, vitamin transport, lipid transport, and carbohydrate metabolic process(Fig. 3H).
Based on the KEGG database for pathway enrichment analysis (the top 20 signal pathways for each comparative item are shown in the figure), In liver samples: For PL1 vs CG1, the pathways significantly enriched with differentially expressed genes include Phagosome, Staphylococcus aureus infection, Leishmaniasis, PPAR signaling pathway, Antigen processing and presentation, Vitamin digestion and absorption, Th17 cell differentiation, Th1 and Th2 cell differentiation, Retinol metabolism, Primary immunodeficiency, AMPK signaling pathway, and Linoleic acid metabolism, etc(Fig. 4A). For PLM1 vs. CG1, the pathways significantly enriched with differentially expressed genes encompass Retinol metabolism, Complement and coagulation cascades, PPAR signaling pathway, Platelet activation, Chemical carcinogenesis - DNA adducts, Steroid hormone biosynthesis, ECM-receptor interaction, AMPK signaling pathway, Biosynthesis of unsaturated fatty acids, and Drug metabolism - cytochrome P450, etc(Fig. 4B). For WT1 vs. CG1, the significantly enriched pathways include the Glucagon signaling pathway, Retinol metabolism, AMPK signaling pathway, Cholesterol metabolism, Arginine biosynthesis, Starch and sucrose metabolism, Glycine, serine and threonine metabolism, Cysteine and methionine metabolism, Fatty acid degradation, PPAR signaling pathway, and ECM-receptor interaction, etc(Fig. 4C). For WTM1 vs. CG1, the significantly enriched pathways are Drug metabolism, Measles, Ascorbate, and alternate metabolism, Pentose and glucuronate interconversions, Influenza A, Porphyrin metabolism, Type I diabetes mellitus, Cholesterol metabolism, Drug metabolism - cytochrome P450, Metabolism of xenobiotics by cytochrome P450, and Chemical carcinogenesis - DNA adducts, etc(Fig. 4D).
In the duodenal samples: For PL2 vs CG2, the pathways significantly enriched with differentially expressed genes are DNA replication, Cell cycle, Staphylococcus aureus infection, Malaria, Viral protein interaction with cytokine and cytokine receptor, IL-17 signaling pathway, Progesterone-mediated oocyte maturation, Complement and coagulation cascades, Pyrimidine metabolism, Neomycin, kanamycin, and gentamicin biosynthesis, and One carbon pool by folate, etc(Fig. 4E). For PLM2 vs CG2, the significantly enriched pathways include Natural killer cell-mediated cytotoxicity, Th1 and Th2 cell differentiation, Th17 cell differentiation, Antigen processing and presentation, Primary immunodeficiency, Hematopoietic cell lineage, T cell receptor signaling pathway, PD-L1 expression and PD-1 checkpoint pathway in cancer, Graft-versus-host disease, and Intestinal immune network for IgA production, etc(Fig. 4F). For WT2 vs CG2, the significantly enriched pathways encompass Amoebiasis, Cell adhesion molecules, AGE-RAGE signaling pathway in diabetic complications, DNA replication, Complement, and coagulation cascades, Chemokine signaling pathway, Protein digestion and absorption, Natural killer cell-mediated cytotoxicity, and ECM-receptor interaction, etc(Fig. 4G). For WTM2 vs CG2, the significantly enriched pathways are DNA replication, NF-kappa B signaling pathway, Primary immunodeficiency, Cell cycle, Alcoholic liver disease, PD-L1 expression and PD-1 checkpoint pathway in cancer, T cell receptor signaling pathway, One carbon pool by folate, and Fat digestion and absorption, etc(Fig. 4H).