Mouse methylome studies SRP254051 Track Settings
 
Post-implantation developmental defects induced by mtDNA mutation derive from altered DNA methylome in oocytes [Oocyte]

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 SRX8003625  CpG methylation  Oocyte / SRX8003625 (CpG methylation)   Schema 
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 SRX8003625  CpG reads  Oocyte / SRX8003625 (CpG reads)   Schema 
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 SRX8003626  PMD  Oocyte / SRX8003626 (PMD)   Schema 
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 SRX8003626  CpG methylation  Oocyte / SRX8003626 (CpG methylation)   Schema 
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 SRX8003626  CpG reads  Oocyte / SRX8003626 (CpG reads)   Schema 
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 SRX8003627  PMD  Oocyte / SRX8003627 (PMD)   Schema 
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 SRX8003627  CpG methylation  Oocyte / SRX8003627 (CpG methylation)   Schema 
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 SRX8003627  CpG reads  Oocyte / SRX8003627 (CpG reads)   Schema 
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 SRX8003628  PMD  Oocyte / SRX8003628 (PMD)   Schema 
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 SRX8003628  AMR  Oocyte / SRX8003628 (AMR)   Schema 
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 SRX8003628  CpG methylation  Oocyte / SRX8003628 (CpG methylation)   Schema 
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 SRX8003628  CpG reads  Oocyte / SRX8003628 (CpG reads)   Schema 
    

Study title: Post-implantation developmental defects induced by mtDNA mutation derive from altered DNA methylome in oocytes
SRA: SRP254051
GEO: GSE147547
Pubmed: not found

Experiment Label Methylation Coverage HMRs HMR size AMRs AMR size PMDs PMD size Conversion Details
SRX8003623 Oocyte 0.346 3.2 9980 37202.9 155 1107.4 3368 320067.9 0.931 title: GSM4433065 D257A ooycte rep1, Mus musculus, Bisulfite-Seq; {"source_name": "D257A treated oocyte", "genotype": "D257A treated"}
SRX8003624 Oocyte 0.348 2.6 7594 38023.4 62 1187.7 2564 395930.3 0.927 title: GSM4433066 D257A ooycte rep2, Mus musculus, Bisulfite-Seq; {"source_name": "D257A treated oocyte", "genotype": "D257A treated"}
SRX8003625 Oocyte 0.342 3.2 8531 41389.3 136 1060.9 3286 338260.6 0.930 title: GSM4433067 D257A ooycte rep3, Mus musculus, Bisulfite-Seq; {"source_name": "D257A treated oocyte", "genotype": "D257A treated"}
SRX8003626 Oocyte 0.387 3.3 7059 43955.5 183 854.1 3160 354176.2 0.920 title: GSM4433068 Wild type oocyte rep1, Mus musculus, Bisulfite-Seq; {"source_name": "Wild Type Oocyte", "genotype": "wild type"}
SRX8003627 Oocyte 0.407 3.4 4540 54646.4 530 919.1 3168 341286.9 0.925 title: GSM4433069 Wild type oocyte rep2, Mus musculus, Bisulfite-Seq; {"source_name": "Wild Type Oocyte", "genotype": "wild type"}
SRX8003628 Oocyte 0.369 3.5 11622 36989.5 164 933.6 3816 294676.7 0.917 title: GSM4433070 Wild type oocyte rep3, Mus musculus, Bisulfite-Seq; {"source_name": "Wild Type Oocyte", "genotype": "wild type"}

Methods

All analysis was done using a bisulfite sequnecing data analysis pipeline DNMTools developed in the Smith lab at USC.

Mapping reads from bisulfite sequencing: Bisulfite treated reads are mapped to the genomes with the abismal program. Input reads are filtered by their quality, and adapter sequences in the 3' end of reads are trimmed. This is done with cutadapt. Uniquely mapped reads with mismatches/indels below given threshold are retained. For pair-end reads, if the two mates overlap, the overlapping part of the mate with lower quality is discarded. After mapping, we use the format command in dnmtools to merge mates for paired-end reads. We use the dnmtools uniq command to randomly select one from multiple reads mapped exactly to the same location. Without random oligos as UMIs, this is our best indication of PCR duplicates.

Estimating methylation levels: After reads are mapped and filtered, the dnmtools counts command is used to obtain read coverage and estimate methylation levels at individual cytosine sites. We count the number of methylated reads (those containing a C) and the number of unmethylated reads (those containing a T) at each nucleotide in a mapped read that corresponds to a cytosine in the reference genome. The methylation level of that cytosine is estimated as the ratio of methylated to total reads covering that cytosine. For cytosines in the symmetric CpG sequence context, reads from the both strands are collapsed to give a single estimate. Very rarely do the levels differ between strands (typically only if there has been a substitution, as in a somatic mutation), and this approach gives a better estimate.

Bisulfite conversion rate: The bisulfite conversion rate for an experiment is estimated with the dnmtools bsrate command, which computes the fraction of successfully converted nucleotides in reads (those read out as Ts) among all nucleotides in the reads mapped that map over cytosines in the reference genome. This is done either using a spike-in (e.g., lambda), the mitochondrial DNA, or the nuclear genome. In the latter case, only non-CpG sites are used. While this latter approach can be impacted by non-CpG cytosine methylation, in practice it never amounts to much.

Identifying hypomethylated regions (HMRs): In most mammalian cells, the majority of the genome has high methylation, and regions of low methylation are typically the interesting features. (This seems to be true for essentially all healthy differentiated cell types, but not cells of very early embryogenesis, various germ cells and precursors, and placental lineage cells.) These are valleys of low methylation are called hypomethylated regions (HMR) for historical reasons. To identify the HMRs, we use the dnmtools hmr command, which uses a statistical model that accounts for both the methylation level fluctations and the varying amounts of data available at each CpG site.

Partially methylated domains: Partially methylated domains are large genomic regions showing partial methylation observed in immortalized cell lines and cancerous cells. The pmd program is used to identify PMDs.

Allele-specific methylation: Allele-Specific methylated regions refers to regions where the parental allele is differentially methylated compared to the maternal allele. The program allelic is used to compute allele-specific methylation score can be computed for each CpG site by testing the linkage between methylation status of adjacent reads, and the program amrfinder is used to identify regions with allele-specific methylation.

For more detailed description of the methods of each step, please refer to the DNMTools documentation.