Mouse methylome studies SRP405928 Track Settings
 
Optimized bisulfite sequencing reveals the lack of 5-methylcytosine in mammalian mitochondrial DNA [WGBS] [Brain, Breast Cancer, ESC, Embryonic Kidney, Hepatocarcinoma, Lung Adenocarcinoma, Melanoma, Neuroblastoma, Platelet]

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 SRX18122815  HMR  Brain / SRX18122815 (HMR)   Schema 
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 SRX18122814  AMR  Melanoma / SRX18122814 (AMR)   Schema 
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 SRX18122814  CpG reads  Melanoma / SRX18122814 (CpG reads)   Schema 
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 SRX18122814  CpG methylation  Melanoma / SRX18122814 (CpG methylation)   Schema 
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 SRX18122814  PMD  Melanoma / SRX18122814 (PMD)   Schema 
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 SRX18122815  AMR  Brain / SRX18122815 (AMR)   Schema 
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 SRX18122815  CpG reads  Brain / SRX18122815 (CpG reads)   Schema 
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 SRX18122815  CpG methylation  Brain / SRX18122815 (CpG methylation)   Schema 
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 SRX18122815  PMD  Brain / SRX18122815 (PMD)   Schema 
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 SRX18122817  HMR  ESC / SRX18122817 (HMR)   Schema 
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 SRX18122816  AMR  Lung Adenocarcinoma / SRX18122816 (AMR)   Schema 
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 SRX18122816  CpG reads  Lung Adenocarcinoma / SRX18122816 (CpG reads)   Schema 
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 SRX18122816  CpG methylation  Lung Adenocarcinoma / SRX18122816 (CpG methylation)   Schema 
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 SRX18122816  PMD  Lung Adenocarcinoma / SRX18122816 (PMD)   Schema 
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 SRX18122817  AMR  ESC / SRX18122817 (AMR)   Schema 
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 SRX18122817  CpG reads  ESC / SRX18122817 (CpG reads)   Schema 
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 SRX18122817  CpG methylation  ESC / SRX18122817 (CpG methylation)   Schema 
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 SRX18122817  PMD  ESC / SRX18122817 (PMD)   Schema 
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 SRX18122818  AMR  ESC / SRX18122818 (AMR)   Schema 
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 SRX18122818  CpG methylation  ESC / SRX18122818 (CpG methylation)   Schema 
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 SRX18122818  PMD  ESC / SRX18122818 (PMD)   Schema 
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 SRX18122818  CpG reads  ESC / SRX18122818 (CpG reads)   Schema 
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 SRX18122819  AMR  Neuroblastoma / SRX18122819 (AMR)   Schema 
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 SRX18122819  CpG reads  Neuroblastoma / SRX18122819 (CpG reads)   Schema 
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 SRX18122819  CpG methylation  Neuroblastoma / SRX18122819 (CpG methylation)   Schema 
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 SRX18122819  PMD  Neuroblastoma / SRX18122819 (PMD)   Schema 
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 SRX18122820  AMR  Neuroblastoma / SRX18122820 (AMR)   Schema 
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 SRX18122820  CpG reads  Neuroblastoma / SRX18122820 (CpG reads)   Schema 
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 SRX18122820  CpG methylation  Neuroblastoma / SRX18122820 (CpG methylation)   Schema 
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 SRX18122820  PMD  Neuroblastoma / SRX18122820 (PMD)   Schema 
    

Study title: Optimized bisulfite sequencing reveals the lack of 5-methylcytosine in mammalian mitochondrial DNA [WGBS]
SRA: SRP405928
GEO: not found
Pubmed: not found

Experiment Label Methylation Coverage HMRs HMR size AMRs AMR size PMDs PMD size Conversion Details
SRX18122814 Melanoma 0.672 11.9 51380 9147.0 3287 2148.4 1876 527302.1 0.993 title: GSM6705301 B16, Mus musculus, Bisulfite-Seq; {"source_name": "melanoma cell", "cell_type": "melanoma cell", "genotype": "WT", "geo_loc_name": "missing", "collection_date": "missing"}
SRX18122815 Brain 0.682 3.9 22147 2012.5 110 2235.0 227 62921.2 0.991 title: GSM6705302 Brain, Mus musculus, Bisulfite-Seq; {"source_name": "mouse brain", "cell_type": "mouse brain", "genotype": "WT", "geo_loc_name": "missing", "collection_date": "missing"}
SRX18122816 Lung Adenocarcinoma 0.616 13.0 49320 7751.7 7490 2139.1 1683 588958.3 0.996 title: GSM6705303 LUAD, Mus musculus, Bisulfite-Seq; {"source_name": "lung adenocarcinoma cell", "cell_type": "lung adenocarcinoma cell", "genotype": "WT", "geo_loc_name": "missing", "collection_date": "missing"}
SRX18122817 ESC 0.595 4.3 29318 1850.1 487 2694.5 353 85584.3 0.992 title: GSM6705304 mESC, Mus musculus, Bisulfite-Seq; {"source_name": "embryonic stem cell", "cell_type": "embryonic stem cell", "genotype": "WT", "geo_loc_name": "missing", "collection_date": "missing"}
SRX18122818 ESC 0.530 3.3 15103 1695.0 1013 1384.5 19 53673.7 0.994 title: GSM6705305 Tet TKO mESC, Mus musculus, Bisulfite-Seq; {"source_name": "embryonic stem cell", "cell_type": "embryonic stem cell", "genotype": "Tet TKO", "geo_loc_name": "missing", "collection_date": "missing"}
SRX18122819 Neuroblastoma 0.360 4.2 10875 15363.1 4256 1494.5 1356 933533.3 0.995 title: GSM6705306 N2a, Mus musculus, Bisulfite-Seq; {"source_name": "neuroblastoma cell", "cell_type": "neuroblastoma cell", "genotype": "WT", "geo_loc_name": "missing", "collection_date": "missing"}
SRX18122820 Neuroblastoma 0.417 3.5 2489 42887.7 165 1908.7 1233 1064903.8 0.994 title: GSM6705307 N2a, mtDNA enriched, Mus musculus, Bisulfite-Seq; {"source_name": "neuroblastoma cell", "cell_type": "neuroblastoma cell", "genotype": "WT", "geo_loc_name": "missing", "collection_date": "missing"}

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.