A Cost Reduced Variant of Epi-Genotyping by Sequencing for Studying DNA Methylation in Non-model Organisms

et al. 2021

Preprint

Background: In fish, minimally invasive blood sampling is widely used to monitor physiological stress with blood plasma biomarkers. As fish blood cells are nucleated, they might be a source a potential new markers derived from ‘omics technologies. We modified the epiGBS (epiGenotyping By Sequencing) technique to explore changes in genome-wide cytosine methylation to a repeated acute stress challenge in the red blood cells (RBCs) of the European sea bass (Dicentrarchus labrax), species is widely studied in both natural and farmed environments.Results: We retrieved 501,108,033 sequencing reads after trimming, with a mean mapping efficiency of 73.0% (unique best hits). Minor changes in RBC methylome appeared to manifest after the challenge test and a family-effect was detected. Fifty-seven differentially methylated cytosines (DMCs) close to 51 distinct genes distributed on 17 of 24 linkage groups (LGs) were detected between RBCs of pre- and post-challenge individuals. Thirty-seven of these genes were previously reported as differentially expressed in the brain of zebrafish, most of them involved in stress coping differences. While further investigation remains necessary, few DMC-related genes associated to the Brain Derived Neurotrophic Factor, a protein that favors stress adaptation and fear memory, appear relevant to integrate a centrally produced stress response in RBCs.Conclusion: Our modified epiGBS protocol was powerful to analyze patterns of cytosine methylation in RBCs of D. labrax and to evaluate the impact of a stress challenge using minimally invasive blood samples. This study is the first approximation to identify epigenetic biomarkers of exposure to stress in fish.

Section: Resultsmentioning

confidence: 79%

Epigenomic response of red blood cells to a stress challenge in the European sea bass (Dicentrarchus labrax, L.)

et al. 2021

Preprint

“…While it should be compared to others (e.g. [104,198,199]), our protocol is close to the double digestion restriction site DNA sequencing protocol used in genomics. Their parallel use certainly deserves attention to design more integrated epigenomic-genomic studies [200,201], and, more generally, multi-omics investigations of stress, health and welfare [202,203].…”

Section: Resultsmentioning

confidence: 99%

Genome-wide epigenomic stress response in the European sea bass (Dicentrarchus labrax, L.)

et al. 2020

Preprint

AbstractUnderstanding the molecular basis of stress is of long standing interest in biology and fish science. We tackled this question by modifying the epiGBS (epiGenotyping By sequencing) technique to screen for cytosine methylation and explore the genome-wide epigenomic response to a repeated acute stress challenge in the European sea bass (Dicentrarchus labrax). Following a minimally invasive sampling using nucleated red blood cells (RBCs), our modified epiGBS protocol retrieved 501,108,033 sequencing reads after trimming, with a mean mapping efficiency of 73.0% (unique best hits). Sequencing reads mapped across all linkage groups (LGs). A total of 47,983 CpG coordinates with a minimum 30X read depth was retained for differential methylation analysis between pre- and post-stress fish. A family effect was demonstrated, and 57 distinct differentially methylated cytosines (DMCs) distributed on 17 of 24 LGs were found between RBCs of pre- and post-stress individuals and located close to 51 distinct stress-related genes. Thirty-eight of these genes were previously reported as differentially expressed in the brain of zebrafish, most of them involved in stress coping differences. Some DMC-related genes appear as good candidates to study the stress response, especially a set of them associated to the Brain Derived Neurotrophic Factor (BDNF), a protein that favors stress adaptation and fear memory. Limits to our study and future directions are presented, including the use of RBCs as a surrogate to other target tissues, to provide with classical physiological measurements a more complete picture of the stress response in fish.

“…While it should be compared to others (e.g. [104,199,200]), our protocol is close to the double digestion restriction site DNA sequencing protocol used in genomics. Their parallel use certainly deserves attention to design more integrated epigenomic-genomic studies [201,202], and multi-omics investigations of stress, health and welfare [203,204].…”

Section: Resultsmentioning

confidence: 99%

Genome-wide epigenomic stress response in the European sea bass (Dicentrarchus labrax, L.)

et al. 2020

Preprint

Background: While the stress response inspired genome-wide epigenetic studies in vertebrate models, it remains mostly ignored in fish. We modified the epiGBS (epiGenotyping By sequencing) technique to explore changes in genome-wide cytosine methylation to a repeated acute stress challenge in the nucleated red blood cells (RBCs) of the European sea bass (Dicentrarchus labrax). This species is widely studied in both the natural and farmed environments, including issues regarding health and welfare.Results: We retrieved 501,108,033 sequencing reads after trimming, with a mean mapping efficiency of 73.0% (unique best hits). Minor changes in RBC methylome appear to manifest after the stress challenge. Only, fifty-seven differentially methylated cytosines (DMCs) close to 51 distinct stress-related genes distributed on 17 of 24 linkage groups (LGs) were detected between RBCs of pre- and post-stress individuals. However, literature surveys indicated that 38 of these genes were previously reported as differentially expressed in the brain of zebrafish, most of them involved in stress coping differences. DMC-related genes associated to the Brain Derived Neurotrophic Factor, a protein that favors stress adaptation and fear memory, appear especially relevant to integrate a centrally produced stress response.Conclusion: By putting forward DMCs associated to stress-related genes, we show that minimally invasive RBCs deserve more attention to investigate the epigenetic response to stress and components of the stress response without sacrificing fish. In parallel to blood parameter measurements (e.g. cortisol, glucose levels), and other molecular approaches (e.g. gene expression variation), features of the epigenetic landscape may offer new opportunities for biomonitoring components of the stress response in fish.