Enzymatic Methyl sequencing of gills and mantle before and after POMS infection in Pacific oyster Magallana gigas GESTINOV 2021

Oyster Sampling and Production Five F1 oyster populations (#15) were produced from progenitors collected from four French oyster production basins in 2022, except population SC18 (#5), maintained under biosecure conditions since 2007. The F1 populations were grown for one year in tanks and fed phytoplankton, with exposure to POMS strictly controlled. POMS Infection and Viral Suspension Two oyster populations (F14 and NSI) were used as donors to generate OsHV-1 Var viral suspensions. Donors were injected with the virus, then cohabited with 100 tagged recipient oysters from the five F1 populations. Infection progression was monitored every two hours, categorizing oysters as susceptible or resistant based on their ability to close their valves. Tissue samples were collected pre- and post-infection and stored for further analysis. Survival and DNA Analysis Survival analysis of both donors and recipients was performed using Kaplan-Meier and Cox proportional hazard models. Genomic DNA was extracted from the gills and mantle, and the viral load of OsHV-1 Var was quantified using qPCR. DNA quality was assessed via nanodrop and Qubit measurements, followed by gel electrophoresis. EM-seq and Bioinformatics Analysis EM-seq libraries were prepared and sequenced, with raw reads processed using FastQC, TrimGalore!, and Bismark for alignment and methylation calling. Differential methylation regions (DMRs) were identified using 'DSS' and assessed for overlap with gene body regions of the M. gigas genome. Conversion efficiency was checked with bacteriophage lambda spike-in controls.

牡蛎采样与繁育：2022年，从法国四个牡蛎养殖流域采集亲本，繁育得到5个F1（子一代）牡蛎种群（编号#15）；其中SC18种群（编号#5）自2007年起即在生物安全可控环境下保种。上述F1种群于水槽中培育一年，投喂浮游植物，且对其接触POMS的过程严格管控。

POMS感染与病毒悬液制备：选取两个牡蛎种群（F14与NSI）作为供体，用以制备OsHV-1 Var病毒悬液。向供体牡蛎注射病毒后，将其与来自上述5个F1种群的100只标记受体牡蛎共同饲养。每两小时监测感染进程，根据牡蛎闭壳能力将其划分为易感型或耐受型。分别于感染前及感染后采集组织样本，保存以备后续分析。

存活分析与DNA检测：采用卡普兰-迈耶（Kaplan-Meier）法与考克斯比例风险（Cox proportional hazard）模型对供体与受体牡蛎开展存活分析。从鳃与外套膜中提取基因组DNA，并通过qPCR（实时荧光定量PCR）定量检测OsHV-1 Var的病毒载量。通过超微量核酸蛋白测定仪（Nanodrop）与Qubit荧光定量系统评估DNA质量，随后进行凝胶电泳检测。

EM-seq与生物信息学分析：构建EM-seq文库并进行测序，原始测序读段通过FastQC、TrimGalore!与Bismark进行质控、序列修剪、基因组比对及甲基化位点识别。采用"DSS"软件识别差异甲基化区域（DMRs），并评估其与太平洋牡蛎（Magallana gigas）基因组基因体区域的重叠情况。通过lambda噬菌体内参对照检测转化效率。