BackgroundGenome-wide profiling of single-nucleotide polymorphisms is receiving increasing attention as a method of pre-implantation genetic diagnosis in humans and of commercial genotyping of pre-transfer embryos in cattle. However, the very small quantity of genomic DNA in biopsy material from early embryos poses daunting technical challenges. A reliable whole-genome amplification (WGA) procedure would greatly facilitate the procedure.ResultsSeveral PCR-based and non-PCR based WGA technologies, namely multiple displacement amplification, quasi-random primed library synthesis followed by PCR, ligation-mediated PCR, and single-primer isothermal amplification were tested in combination with different DNA extractions protocols for various quantities of genomic DNA inputs. The efficiency of each method was evaluated by comparing the genotypes obtained from 15 cultured cells (representative of an embryonic biopsy) to unamplified reference gDNA. The gDNA input, gDNA extraction method and amplification technology were all found to be critical for successful genome-wide genotyping. The selected WGA platform was then tested on embryo biopsies (n = 226), comparing their results to that of biopsies collected after birth. Although WGA inevitably leads to a random loss of information and to the introduction of erroneous genotypes, following genomic imputation the resulting genetic index of both sources of DNA were highly correlated (r = 0.99, P<0.001).ConclusionIt is possible to generate high-quality DNA in sufficient quantities for successful genome-wide genotyping starting from an early embryo biopsy. However, imputation from parental and population genotypes is a requirement for completing and correcting genotypic data. Judicious selection of the WGA platform, careful handling of the samples and genomic imputation together, make it possible to perform extremely reliable genomic evaluations for pre-transfer embryos.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-889) contains supplementary material, which is available to authorized users.

Successful somatic cell nuclear transfer (SCNT) requires epigenetic reprogramming of a differentiated donor cell nucleus. Incorrect reprogramming of epigenetic markings such as DNA methylation is associated with compromised prenatal development and postnatal abnormalities. Clones that survive into adulthood, in contrast, are assumed to possess a normalized epigenome corresponding to their normal phenotype. To address this point, we used capillary electrophoresis to measure 5-methylcytosine (5mC) levels in leukocyte DNA of 38 healthy female bovine clones that represented five genotypes from the Simmental breed and four genotypes from the Holstein breed. The estimated variance in 5mC level within clone genotypes of both breeds [0.104, 95% confidence interval (CI): 0.070-0.168] was higher than between clone genotypes (0, CI: 0-0.047). We quantified the contribution of SCNT to this unexpected variability by comparing the 19 Simmental clones with 12 female Simmental monozygotic twin pairs of similar age. In Simmental clones, the estimated variability within genotype (0.0636, CI: 0.0358-0.127) was clearly higher than in twin pairs (0.0091, CI: 0.0047-0.0229). In clones, variability within genotype (0.0636) was again higher than between genotypes (0, CI: 0-0.077). Twins, in contrast, showed lower variability within genotypes (0.0091) than between genotypes (0.0136, CI: 0.00250-0.0428). Importantly, the absolute deviations of 5mC values of individual SCNT clones from their genotype means were fivefold increased in comparison to twins. Further comparisons with noncloned controls revealed DNA hypermethylation in most of the clones. The clone-specific variability in DNA methylation and DNA hypermethylation clearly show that healthy adult SCNT clones must be considered as epigenome variants.

BackgroundIt was recently established that changes in methylation during development are dynamic and involve both methylation and demethylation processes. Yet, which genomic sites are changing and what are the contributions of methylation (5mC) and hydroxymethylation (5hmC) to this epigenetic remodeling is still unknown. When studying early development, options for methylation profiling are limited by the unavailability of sufficient DNA material from these scarce samples and limitations are aggravated in non-model species due to the lack of technological platforms. We therefore sought to obtain a representation of differentially 5mC or 5hmC loci during bovine early embryo stages through the use of three complementary methods, based on selective methyl-sensitive restriction and enrichment by ligation-mediated PCR or on subtractive hybridization. Using these strategies, libraries of putative methylation and hydroxymethylated sites were generated from Day-7 and Day-12 bovine embryos.ResultsOver 1.2 million sequencing reads were analyzed, resulting in 151,501 contigs, of which 69,136 were uniquely positioned on the genome. A total of 101,461 putative methylated sites were identified. The output of the three methods differed in genomic coverage as well as in the nature of the identified sites. The classical MspI/HpaII combination of restriction enzymes targeted CpG islands whereas the other methods covered 5mC and 5hmC sites outside of these regions. Data analysis suggests a transition of these methylation marks between Day-7 and Day-12 embryos in specific classes of repeat-containing elements.ConclusionsOur combined strategy offers a genomic map of the distribution of cytosine methylation/hydroxymethylation during early bovine embryo development. These results support the hypothesis of a regulatory phase of hypomethylation in repeat sequences during early embryogenesis.

Cloning by nuclear transfer has made it possible to produce genetically identical animals in terms of nuclear DNA content. Recent molecular biology tools are offering scientific ways to get an insight into the identity issues, by exploring and comparing genomes of cloned animals in order to test their genetic identity and methylation differences. We have initiated a study to compare genomic DNA of bovine adult clones, of normal phenotype. We have used, in parallel, the AFLP technique (amplification fragment length polymorphism) and one of its variant, MSAP (methylation-sensitive amplification polymorphism). We are also investigating other techniques leading to the detection of sequence polymorphisms between two genomes based on genomes hybridisation. We chose the representational difference analysis (RDA) methods that can be combined with mismatch-specific recognition or mismatch binding property of some proteins (CEL I, MutS). We plan to use these RDA methods for genome-wide detection of subtle mutations, then to focus on changes affecting the methylation status of promoting genomic regions in abnormal clones. This will be achieved using MSAP with NotI and applying, in parallel, the RLGS (restriction landmark genome scanning) technique. This study will hopefully improve the molecular and functional characterizations of these "new animals."

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