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| Status |
Public on Oct 01, 2023 |
| Title |
K_AB_rep2 |
| Sample type |
SRA |
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| Source name |
head kidney
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| Organism |
Abramis brama |
| Characteristics |
tissue: head kidney
treatment: K
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| Treatment protocol |
OK experiment was performed using twenty-five larval (oncomiracidia) specimens of P. homoion that were transferred into the aerated and standing tap water in each aquarium in which a single naïve fish specimen of five fish lines (R. rutilus, A. brama, F1 generation with A. brama mtDNA, backcross generation of F1 hybrid with A. brama mtDNA and paternal A. brama, backcross generation of F1 hybrid with A. brama mtDNA and paternal R. rutilus) was placed.
PH experiment was performed as cohabitation experiment. A total of eight fish specimens per each of six fish lines (A. brama, R. rutilus, F1 generation with A. brama mtDNA, F1 generation with R. rutilus mtDNA, backcross generation of F1 hybrid with A. brama mtDNA and paternal A. brama, backcross generation of F1 hybrid with A. brama mtDNA and paternal R. rutilus) were all placed in one tank with aerated water under temperature 20°C together with 25 specimens of P. homoion-infected donor fish Rhodeus ocellatus (Asian species was used in the laboratory to retain life cycle of P. homoion).
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| Growth protocol |
Specimens of A. brama, R. rutilus, and their F1 hybrids were collected from the Hamry Reservoir (49.73724N, 15.91395E; the Czech Republic) and transported to the breeding facility. The hybrids were identified using meristic traits (i.e., the number of gill rakers, the number of scales in the lateral line, and the number of branched rays in the anal fin) and molecular markers (the cytochrome b gene and microsatellite loci). The fish were separated according to sex into two well-aerated tanks and stimulated for ovulation/spermiation by carp pituitary (females received 2 doses, 0.3 and 2.7 mg/kg, 24 h and 12 h before propagation, respectively; males received 1 mg/kg 24 h before propagation) and by subsequently increasing the water temperature to 22 °C. Oocytes of ovulating females were obtained by the dry method and sperm was sampled. Hatchery water was used for gamete activation. Artificial spawning based on individual pair mating was performed using the following parental combinations: (1) A. brama female and A. brama male (2) R. rutilus female and R. rutilus male, (3) A. brama female and R. rutilus male, (4) A. brama male and R. rutilus female, (5) F1 hybrid female (with A. brama mtDNA) and A. brama male, and (6) F1 hybrid female (with A. brama mtDNA) and R. rutilus male. As a result of artificial crosses, one R. rutilus line, one A. brama line, one F1 hybrid line with A. brama mtDNA (termed F1 A. brama x R. rutilus), one F1 hybrid line with R. rutilus mtDNA (termed F1 R. rutilus x A. brama), and two backcross lines, i.e. the backcross generation resulting from the crosses of both parents with A. brama mtDNA (F1 hybrid female with A. brama mtDNA and A. brama male, termed BC of F1 hybrid x A. brama), and the backcross generation resulting from the crosses of parents with different mtDNA (F1 hybrid female with A. brama mtDNA and R. rutilus male, termed BC of F1 hybrid x R. rutilus). All fish were reared to the age of 2 years.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Fish were investigated for the presence of parasites one month after oncomiracidia infection (or following cohabitation) by fish dissection, and parasites were counted. Head kidney was collected from individual fish including 4-5 non-infected control specimens per each fish line and at least 4 P. homoion-infected specimens per each of five fish lines (R. rutilus, A. brama, F1 generation with A. brama mtDNA, backcross generation of F1 hybrid with A. brama mtDNA and paternal A. brama, backcross generation of F1 hybrid with A. brama mtDNA and paternal R. rutilus) in the case of OK experiment, six fish lines in the case of PH experiment including F1 generation with R. rutilus mtDNA.
Total RNA was isolated from the head kidney 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 (15.3 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 analyzed using RNA 6000 Nano Kit on a 2100 Bioanalyzer 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 and for the reverse transcription of total RNA into single-stranded cDNA using High Capacity RNA-to-cDNA Kit (Applied Biosystems by Thermo Fisher Scientific) in twice the reaction volume recommended by the manufacturer.
All fish samples (RNA integrity number – RIN > 7) were used for DNA library preparation. 500 ng 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 Bioanalyzer with DNA 1000 Kit (Agilent Technologies). Finally, amplicons were pooled in equimolar amounts. The final concentration of each particular library in the pool was 10nM in the pool. Libraries were sequenced by Macrogen Europe B.V. (Amsterdam, Netherlands) on an Illumina NovaSeq6000 system (one line on an S4 flow cell) 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 |
Illumina NovaSeq 6000 |
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| Description |
Abramis brama
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| Data processing |
A quality check of raw paired-end fastq reads was carried out by FastQC and a contamination check against human (GRCh38), mouse (GRCm38), yeast (S. cerevisiae R64-1-1), E. coli BL21(DE3) and other organisms by BioBloom tools.
The quality and Illumina adapter trimming of raw reads was performed using Trimmomatic v0.39 (options: PE CROP:250 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:5 ILLUMINACLIP:adapters.fa:2:30:10:3:true MINLEN:35).
Trimmed reads were aligned to the reference transcriptome using STAR v2.7.3a with options: --runMode alignReads --sjdbOverhang 100 --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax 1.0 --outFilterMismatchNoverLmax 0.1 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outFilterMatchNmin 0 --outFilterScoreMinOverLread 0.66 --outFilterMatchNminOverLread 0.66 --outSAMheaderHD @HD VN:1.4 SO:coordinate --peOverlapMMp 0.1 --chimOutJunctionFormat 1 --chimSegmentMin 12 --chimJunctionOverhangMin 12 --chimOutType Junctions SeparateSAMold --outSAMunmapped Within --outFilterType BySJout --outSAMattributes All --quantMode GeneCounts TranscriptomeSAM --sjdbScore 1 --twopassMode Basic --outMultimapperOrder Random --outSAMtype BAM SortedByCoordinate
The mapped reads were deduplicated by Picard’s MarkDuplicates v2.27.1 with options: -REMOVE_DUPLICATES False -ASSUME_SORTED true -PROGRAM_RECORD_ID null -VALIDATION_STRINGENCY LENIENT
The quantification of gene expression was performed by Subread's featureCounts v1.6.4 with options: -t exon -g gene_id -p -P -C -s 0 -T 10 -F GTF -Q 0 -d 1 -D 25000
Differential gene expression analysis between infected and non-infected individuals for each fish line of OK experiment was performed according to gene counts produced by featureCounts, and further processed using the Bioconductor package DESeq2 v1.34.0. Genes were considered as differentially expressed only if having the adjusted p-value (FDR) smaller than 0.05 and the absolute value of the log2 expression fold-change higher than 1. The DESeq2 tool computes the normalized and shrunk log2 expression fold-change for each gene (for the results see supplemetray files called deseq2...tsv).
The functional role of significantly differentially expressed genes (i.e., enrichment analysis) was performed according to the KEGG pathway database and the Gene ontology database. For each fish line, the differentially expressed genes were used as the target set, and all of the annotated genes within the transcriptome served as a background for the statistical testing. The core of the analysis was performed by an in-house script using the R package clusterProfiler for individual fish lines separately for three sets: up-, down- and dis-regulated significantly differentially expressed genes. The Gene ontology (GO) annotation comes from the transcriptome assembly of R. rutilus using the semi-automatic annotation tool called Trinotate. From the total of 33767 predicted genes, 12590 were annotated with GO. KEGG annotation comes from the KAAS - KEGG Automatic Annotation Server using GHOSTZ on peptide sequences of predicted genes against the set of all fish genes of the KEGG database. From the total of 33767 predicted genes, 12618 were annotated with KEGG orthology identifiers (for the results see supplemetray files called GO_enrich...tsv and KEGG_enrich...tsv, respectively).
Assembly: R. rutilus transcriptome assembly
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample
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| Submission date |
Aug 15, 2023 |
| Last update date |
Oct 01, 2023 |
| 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 |
GPL33681 |
| Series (1) |
| GSE240906 |
Transcriptome profile analyses of head kidney in roach (Rutilus rutilus), common bream (Abramis brama) and their hybrids: does infection by monogenean parasites in freshwater fish reveal differences in fish vigour among parental species and their hybrids? |
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| Relations |
| BioSample |
SAMN36995807 |
| SRA |
SRX21374944 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7711775_K_AB_rep2.featureCounts.tsv.gz |
647.7 Kb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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