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Sample GSM1521874

Query DataSets for GSM1521874

Status

Public on Jun 18, 2015

Title

Smolt_Female_3C59

Sample type

SRA

Source name

caudal fin tissue

Organism

Oncorhynchus mykiss

Characteristics

tissue type: caudal fin
migration-related phenotype: Smolt
Sex: Female

Treatment protocol

When individuals reached age 1+ or 2+ years old (the temporal period when smoltification is likely to occur), multiple phenotypes strongly associated with smoltification (and thereby migration) were measured after fish were anesthetized with clove oil. These included body morphology, skin reflectance, gill Na+/K+ ATPase activity, body condition factor, and growth rates. Measurements from these phenotypes were compiled together to create a binary phenotype: migrant or resident trout.

Growth protocol

Migratory steelhead (Clearwater) and resident rainbow trout (Oregon State University) double haploid clonal lines, which are each completely homozygous, were crossed to create F1 hybrids. Gametes from an F1 male then underwent androgenesis, to create F2 progeny, each being homozygous across loci, but genetically different from their siblings due to meiotic recombination. Doubled haploids were produced in October and reared for ~3 years at the Washington State University research hatchery. Embryos were maintained in recirculating stack egg incubators at a constant 11° until complete yolk absorption. Fish were then transferred to 5-gallon tanks maintained in a recirculating system at similar densities. At 6 months of age, all fish were transferred to a single 200-gallon tank and were fed by automatic feeder, which was replenished once daily. Temperatures in the recirculating systems ranged from 11° to 18° throughout this study.

Extracted molecule

genomic DNA

Extraction protocol

DNA was freshly extracted from caudal fin tissue using the Qiagen DNeasy kit according to the manufacturer's suggested protocol.
RRBS libraries were constructed using a previously published protocol (Boyle et al. 2012, Genome Biology). Briefly, 100 ng genomic DNA was digested using the restriction enzyme MspI. DNA fragments were end-repaired, A-tailed, and ligated to the methylated Illumina TruSeq adapters. Ten samples were multiplexed per library with each sample ligated to unique barcoded Illumina adapters to distinguish samples. Life history types and sex ratios were primarily equalized across libraries. Bisulfite conversion was conducted using Qiagen's EpiTect Bisulfite kit, following the protocol for formalin-fixed paraffin-embedded samples. After two rounds of bisulfite conversion, the DNA was amplified using Illumina TruSeq primers. The optimal number of PCR cycles, in this case 18, was determined empirically by comparing PCR products on a 5% Metaphor agarose gel for 10, 13, 16, 19, and 22 total cycles. Library size selection was gel-free and occurred by using AMPure XP beads as suggested by Boyle et al. 2012. Library DNA quantity was assessed with the Qubit fluorometer and quality was assessed with a 5% MetaPhor agarose gel (Lonza). All sequencing was performed by the Vincent J. Coates Genomics Sequencing Laboratory at the University of California, Berkeley. After a quantitative PCR quality control check, the Illumina HiSeq 2000 platform was used to sequence 50 bp single-end reads for all 20 samples. The mRRBS protocol was repeated for nine samples with fewer than 10 million reads after quality filtering. Read length was increased for these samples up to 100 bp single-end reads. Two of the smolt (migrant) samples had insufficient reads (<10 million reads) and were removed from subsequent analysis. Therefore, 8 smolts were compared to 10 residents.

Library strategy

Bisulfite-Seq

Library source

genomic

Library selection

Reduced Representation

Instrument model

Illumina HiSeq 2000

Description

Processed data shows differentially methylated cytosines (DMCs) and differentially methylated regions (DMRs) when aligning reads to the O. mykiss reference genomes (Miller and French)

Data processing

Illumina CASAVA 1.8.0 used for basecalling
Trim Galore! used to remove adapter sequences and low quality bases from reads (-rrbs option)
Bismark used to map reads onto bisulfite converted reference O. mykiss genomes. Bismark's default parameters were used and only two non-bisulfite mismatches were allowed per read.
methylKit R package used to normalize reads between samples and identify differentially methylated cytosines (DMCs) between migrant and resident groups (default parameters used).
eDMR R package used to identify differentially methylated regions (DMRs) between migrant and resident groups (default parameters used).
Genome_build: Two reference genomes were used since O. mykiss has only been recently sequenced and neither genome is well assembled. Details about one (herein termed the O. mykiss French genome) are available in a publication (Berthelot et al., 2014, Nature Communications) and the genome sequence is in the European Nucleotide Archive under the study PRJEB4421. The other genome draft (herein termed the O. mykiss Miller genome) is available in an online repository (http://www.animalgenome.org/repository/aquaculture/).
Supplementary_files_format_and_content: Processed data files include outputs from: 1) methylKit (differentially methylated cytosines), 2) eDMR (differentially methylated regions); 3) gene annotations for DMRs. Much of the annotation occurred manually [e.g., BLAST searches from Miller genome; searching the Genomicus server (http://www.genomicus.biologie.ens.fr/genomicus-trout-01.01/) for gene names for the French genome].

Submission date

Oct 09, 2014

Last update date

May 15, 2019

Contact name

Melinda Baerwald

E-mail(s)

[email protected]

Organization name

University of California-Davis

Street address

1 Shields Avenue