Unknown,Transcriptomics,Genomics,Proteomics

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Evolutionary consequences of DNA methylation on the GC content in vertebrate genomes

ABSTRACT: The genomes of many vertebrates show a characteristic variation in GC content. To explain its origin and evolution mainly three mechanisms have been proposed, selection for GC content, mutation bias and GC-biased gene conversion. At present the mechanism of GC-biased gene conversion, i.e. short-scale, unidirectional exchanges between homologous chromosomes in the neighborhood of recombination-initiating double-strand breaks in favor for GC nucleotides, is the most widely accepted hypothesis. We here suggest that DNA methylation also plays an important role in the evolution of GC content in vertebrate genomes. To test this hypothesis we investigated one mammalian (human; GSE30340) and one avian (chicken) genome. We used bisulfite sequencing to generate a whole-genome methylation map of chicken sperm. Human processed data files (spermdonor1, #reads>=1) were downloaded from the NGSmethDB database (http://bioinfo2.ugr.es/NGSmethDB/database.php). Inclusion of these methylation maps into a model of GC content evolution provided significant support for the impact of DNA methylation on the local equilibrium GC content. Moreover, two different estimates of equilibrium GC content, one which neglects and one which incorporates the impact of DNA methylation and the concomitant CpG hypermutability, give estimates that differ about 15% in both genomes, arguing for a strong impact of DNA methylation on the evolution of GC content. Thus, our results put forward that previous estimates of equilibrium GC content, which neglect the hypermutability of CpG dinucleotides, need to be reevaluated. Genomic DNA from chicken mature sperm was isolated, bisulfite converted and sequenced on a Illumina HiSeq instrument

ORGANISM(S): Gallus gallus

SUBMITTER: Hans Ellegren

PROVIDER: E-GEOD-56639 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:All large-scale LC-MS/MS post-translational methylation site discovery experiments require methylpeptide spectrum matches (methyl-PSMs) to be identified at acceptably low false discovery rates (FDRs). To meet estimated methyl-PSM FDRs, methyl-PSM filtering criteria are often determined using the target-decoy approach. The efficacy of this methyl-PSM filtering approach has, however, yet to be thoroughly evaluated. Here we conduct a systematic analysis of methyl-PSM FDRs across a range of sample preparation workflows (each differing in their exposure to the alcohols methanol and isopropanol) and mass spectrometric instrument platforms (each employing a different mode of MS/MS dissociation). Through 13CD3-methionine labeling (heavy-methyl SILAC) of S. cerevisiae cells and in-depth manual data inspection, accurate lists of true positive methyl-PSMs were determined, allowing methyl-PSM FDRs to be compared to target-decoy approach-derived methyl-PSM FDR estimates. These results show that global FDR estimates produce extremely unreliable methyl-PSM filtering criteria; we demonstrate that this is an unavoidable consequence of the high number of amino acid combinations capable of producing peptide sequences that are isobaric to methylated peptides of a different sequence. Separate methyl-PSM FDR estimates were also found to be unreliable due to prevalent sources of false positive methyl-PSMs that produce high peptide identity score distributions. Incorrect methylation site localizations, peptides containing cysteinyl-S-β-propionamide, and methylated glutamic or aspartic acid residues can partially, but not wholly, account for these false positive methyl-PSMs. Together these results indicate that the target-decoy approach is an unreliable means of estimating methyl-PSM FDRs and methyl-PSM filtering criteria. We suggest that orthogonal methylpeptide validation (e.g. heavy-methyl SILAC or its offshoots) should be considered a prerequisite for obtaining high confidence methyl-PSMs in large-scale LC-MS/MS methylation site discovery experiments, and make recommendations on how to reduce methyl-PSM FDRs in samples not amenable to heavy isotope labeling.

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Evolutionary consequences of DNA methylation on the GC content in vertebrate genomes

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