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| Status |
Public on May 17, 2024 |
| Title |
C. gibelio, diploid, male, replicate_2 |
| Sample type |
SRA |
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| Source name |
gonadal
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| Organism |
Carassius gibelio |
| Characteristics |
tissue: gonadal
ploidy: diploid
Sex: male
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| Treatment protocol |
Gonadal tissues of each fish specimen were individually submerged in Ambion RNAlater stabilization solution (Thermo Fisher Scientific). Tubes with tissues were stored at -80ºC until the isolation of total RNA.
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| Growth protocol |
Asexual and sexual C. gibelio were obtained from artificial breeding. Asexual females were obtained by induced embryogenesis using sperm of C. carpio. The sexual specimens were obtained from the interbreeding of sexual specimens. The fish were reared in aquarium conditions until the age of four years and subsequently their gonadal tissues were sampled (the age of the examined fish corresponded to 4+). Cyprinus carpio and Carassius auratus were obtained from external breeding facilities. Four or five biological samples per fish group from a total of 8 fish groups were analysed (females and males of C. gibelio resulting from sexual reproduction, females and temperature-induced males of C. gibelio resulting from gynogenesis, females and males of sexual C. auratus, and females and males of sexual C. carpio).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from the gonad tissue of each fish specimen. For extraction, PureLink® RNA Mini Kit (Ambion) with Trizol reagent (Thermo Fisher Scientific) and on-column PureLink DNase treatment were used according to the manufacturer´s protocol. Reagent and buffer volumes were adjusted according to the weight of tissue entering the isolation process (30 mg on average). The final elution was performed using 100 µl of RNAse-free water in the first step and the primal eluate in the second step. The yield and concentration of RNA isolates were checked using a QubitTM 4 fluorometer (Invitrogen by Thermo Fisher Scientific) and Qubit RNA HS Assay Kit (Thermo Fisher Scientific). The quality and integrity of RNA were analysed using RNA 6000 Nano Kit on a 2100 Bioanalyser instrument (Agilent Technologies). All RNA isolates were normalized by dilution at a uniform concentration of 20 ng/µl with RNase-free water. They served as templates for DNA library preparation in twice the reaction volume recommended by the manufacturer.
All samples (RNA integrity number – RIN > 7) were used for DNA library preparation. 500ng of total RNA was used for mRNA enrichment using the Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). Subsequently, NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®, and NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 2, New England Biolabs) were used for library preparation, with 11 PCR cycles utilized for PCR enrichment. RNA fragmentation (13 minutes at 94°C) and the size selection conditions (a bead volume of 30 µl and 15 µl for the first and second bead selections, respectively) were further modified in the protocol. The quantification of DNA libraries was performed on a QubitTM 4 fluorometer (Invitrogen by Thermo Fisher Scientific) using Qubit dsDNA HS Assay Kit, and quality and size control were performed on a 2100 Bioanalyser with DNA 1000 Kit (Agilent Technologies). Finally, amplicons were pooled in equimolar amounts. The final concentration of each library in the pool was 10 nM in the pool. Subsequently, NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® and NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 2, New England Biolabs) together with spike-in RNA were used for cDNA library preparation from total RNA. The quality of prepared cDNA libraries was evaluated using a Qubit fluorometer (Thermo Fisher). The quality of cDNA libraries was visualized by a 2100 Bioanalyser (Agilent), and the libraries were were finally sequenced by Macrogen Korea on Illumina HiSeq X (one lane) in a paired-end configuration producing 150 bp long reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
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| Data processing |
A quality check of raw paired-end fastq reads was carried out by FastQC and their origin was categorized using BioBloomTools v2.3.4. The Illumina adapters clipping and quality trimming of raw fastq reads were performed using Trimmomatic v0.39 with settings CROP:250 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:5 MINLEN:35. Trimmed RNA-Seq reads were mapped to the reference genome using STAR v2.7.3a as a splice-aware short read aligner and default parameters except for --outFilterMismatchNoverLmax 0.1 and --twopassMode Basic. Quality control after alignment concerning the number and percentage of uniquely- and multi-mapped reads, rRNA contamination, mapped regions, read coverage distribution, strand specificity, gene biotypes, and PCR duplication was performed using several tools – namely, RSeQC v4.0.0, Picard toolkit v2.25.6, and Qualimap v.2.2.2. All statistics were processed by MultiQC v1.10.1.
The genomic sequences of all collected samples were aligned to the reference genome utilizing the Burrows-Wheeler Aligner (BWA) software. Post alignment and germline variants were called using Strelka2 variant calling software, generating variant calls in VCF format which were further filtered to retain only high-confidence variants. These variants were then annotated using the reference annotation in GTF file. Subsequent data processing was carried out in R, where the variant tables were further refined and merged with sample information. A series of filtering steps were performed to ensure only variants with sufficient coverage and sample counts were retained for analysis. The filtered variant table was then reorganized and formatted for subsequent comparative analyses. Variants located on sex chromosomes were excluded for certain analyses to ensure accurate cross-species comparisons. The data were then restructured to compare SNP identity across species, generating similarity matrices and Venn diagrams to visualize the overlap of SNPs by species and ploidy levels.
The differential gene expression was calculated on the basis of the gene counts produced using featureCounts from the Subread package v2.0 and further analysed by Bioconductor package DESeq2 v1.34.0. Data generated by DESeq2 with independent filtering were selected for differential gene expression analysis to avoid potential false positive results. Differences in gene expression were considered significant on the basis of a cut-off of the adjusted p-value ≤ 0.05. GO term enrichment was analysed using David to retrieve Gene Ontology terms in the Biological process, Cellular Component and Molecular function categories, as well as KEGG pathways. Graphical representations of the GO enrichment were realized using R and Revigo. Reproduction-associated candidate genes were retrieved using the BlastKoala tool of KEGG, the BioMart tool of Ensembl, and published studies. GO terms enrichment was tested using Fisher’s exact test (α = 0.05) with false discovery rate (FDR) correction of the p-value. To interpret the biological functions of the DEGs, their mapping to the Gene Ontology (GO) and KEGG databases was performed to analyse pathway enrichment. In each of six fish groups associated with sexual reproduction and asexual males, significantly differently-expressed genes (DEGs) compared to the triploid asexual females of C. gibelio were selected on the basis of the following criteria: Basemean > 10, and a padj value < 0.05,. For KEGG pathway analysis, no filtering based on log2 fold change was applied. Gene functions were investigated using the biological databases Uniprot, KEGG, Zfin and GeneCards. PCA was performed using the DESeq2 R package. For PCA based on reproduction-associated genes, a set of 208 reproduction genes was selected using the BioMart tool of Ensembl.
Assembly: Carassius auratus genome (ASM336829v1) with Ensembl annotation (release 104)
Supplementary files format and content: tab-delimited text file includes raw counts for all Samples
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| Submission date |
Jan 23, 2024 |
| Last update date |
May 17, 2024 |
| Contact name |
Andrea Vetešníková Šimková |
| E-mail(s) |
[email protected]
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| Organization name |
Masaryk University
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| Department |
Department of Botany and Zoology
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| Lab |
Parasitology
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| Street address |
Kamenice 5
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| City |
Brno |
| ZIP/Postal code |
62500 |
| Country |
Czech Republic |
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| Platform ID |
GPL34122 |
| Series (1) |
| GSE254010 |
Reproduction-associated pathways in females of gibel carp (Carassius gibelio) shed light on the molecular mechanisms of the coexistence of asexual and sexual reproduction |
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| Relations |
| BioSample |
SAMN39578563 |
| SRA |
SRX23366571 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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