Mouse methylome studies SRP423445 Track Settings
 
Ultrafast Bisulfite Sequencing for Efficient and Accurate 5-Methylcytosine Detection in DNA and RNA [Plasma, Stem Cell, mESC]

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 SRX19430718  CpG methylation  mESC / SRX19430718 (CpG methylation)   Data format 
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 SRX19430721  CpG methylation  mESC / SRX19430721 (CpG methylation)   Data format 
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 SRX19430722  CpG methylation  mESC / SRX19430722 (CpG methylation)   Data format 
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 SRX19430723  CpG methylation  mESC / SRX19430723 (CpG methylation)   Data format 
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 SRX19430727  CpG methylation  mESC / SRX19430727 (CpG methylation)   Data format 
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 SRX19430728  CpG methylation  mESC / SRX19430728 (CpG methylation)   Data format 
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 SRX19430729  CpG methylation  mESC / SRX19430729 (CpG methylation)   Data format 
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 SRX22085402  CpG methylation  Stem Cell / SRX22085402 (CpG methylation)   Data format 
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 SRX22085403  CpG methylation  Stem Cell / SRX22085403 (CpG methylation)   Data format 
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 SRX22085404  CpG methylation  Stem Cell / SRX22085404 (CpG methylation)   Data format 
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 SRX22085405  CpG methylation  Stem Cell / SRX22085405 (CpG methylation)   Data format 
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 SRX22085406  CpG methylation  Stem Cell / SRX22085406 (CpG methylation)   Data format 
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 SRX22085407  CpG methylation  Stem Cell / SRX22085407 (CpG methylation)   Data format 
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 SRX22085408  CpG methylation  Stem Cell / SRX22085408 (CpG methylation)   Data format 
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 SRX22085409  CpG methylation  Stem Cell / SRX22085409 (CpG methylation)   Data format 
    
Assembly: Mouse Jun. 2020 (GRCm39/mm39)

Study title: Ultrafast Bisulfite Sequencing for Efficient and Accurate 5-Methylcytosine Detection in DNA and RNA
SRA: SRP423445
GEO: not found
Pubmed: not found

Experiment Label Methylation Coverage HMRs HMR size AMRs AMR size PMDs PMD size Conversion Title
SRX19430718 mESC 0.189 6.3 1 838820.0 31 1130.1 1 109678624.0 0.991 GSM7051124: 100 mES cells treated with conventional BS, replicate 1; Mus musculus; Bisulfite-Seq
SRX19430719 mESC 0.235 8.2 0 0.0 79 1011.8 0 0.0 0.988 GSM7051125: 100 mES cells treated with conventional BS, replicate 2; Mus musculus; Bisulfite-Seq
SRX19430720 mESC 0.202 8.4 0 0.0 58 1026.4 0 0.0 0.990 GSM7051126: 100 mES cells treated with conventional BS, replicate 3; Mus musculus; Bisulfite-Seq
SRX19430721 mESC 0.236 4.0 1 597931.0 95 964.8 0 0.0 0.989 GSM7051127: 10 mES cells treated with conventional BS, replicate 1; Mus musculus; Bisulfite-Seq
SRX19430722 mESC 0.186 4.2 1 519112.0 132 927.9 1 109670014.0 0.990 GSM7051128: 10 mES cells treated with conventional BS, replicate 2; Mus musculus; Bisulfite-Seq
SRX19430723 mESC 0.210 5.9 1 795055.0 251 929.6 0 0.0 0.991 GSM7051129: 10 mES cells treated with conventional BS, replicate 3; Mus musculus; Bisulfite-Seq
SRX19430727 mESC 0.181 5.8 3 878717.3 7 695.7 0 0.0 0.995 GSM7051133: 100 mES cells treated with ultrafast BS, replicate 1; Mus musculus; OTHER
SRX19430728 mESC 0.166 6.0 1 838820.0 10 762.0 1 118820859.0 0.995 GSM7051134: 100 mES cells treated with ultrafast BS, replicate 2; Mus musculus; OTHER
SRX19430729 mESC 0.193 5.4 1 838820.0 4 1085.0 0 0.0 0.995 GSM7051135: 100 mES cells treated with ultrafast BS, replicate 3; Mus musculus; OTHER
SRX22085402 Stem Cell 0.337 19.7 45986 4527.4 59 945.9 3908 113017.7 0.986 GSM7840492: 10 ng mESC genomic DNA treated with conventional BS replicate 1; Mus musculus; Bisulfite-Seq
SRX22085403 Stem Cell 0.336 15.9 44417 4593.1 63 814.8 3759 113048.7 0.986 GSM7840493: 10 ng mESC genomic DNA treated with conventional BS replicate 2; Mus musculus; Bisulfite-Seq
SRX22085404 Stem Cell 0.346 3.0 1 1145988.0 39 957.4 0 0.0 0.976 GSM7840494: 1 ng mESC genomic DNA treated with conventional BS replicate 1; Mus musculus; Bisulfite-Seq
SRX22085405 Stem Cell 0.340 2.9 1 795486.0 62 965.7 0 0.0 0.978 GSM7840495: 1 ng mESC genomic DNA treated with conventional BS replicate 2; Mus musculus; Bisulfite-Seq
SRX22085406 Stem Cell 0.289 17.4 43818 4619.2 28 760.1 139 774455.0 0.993 GSM7840496: 10 ng mESC genomic DNA treated with ultrafast BS replicate 1; Mus musculus; Bisulfite-Seq
SRX22085407 Stem Cell 0.290 12.0 37226 5317.7 19 849.2 19 1295364.8 0.992 GSM7840497: 10 ng mESC genomic DNA treated with ultrafast BS replicate 2; Mus musculus; Bisulfite-Seq
SRX22085408 Stem Cell 0.290 3.2 1 1354581.0 18 1022.4 0 0.0 0.990 GSM7840498: 1 ng mESC genomic DNA treated with ultrafast BS replicate 1; Mus musculus; Bisulfite-Seq
SRX22085409 Stem Cell 0.289 3.7 0 0.0 41 1106.5 0 0.0 0.989 GSM7840499: 1 ng mESC genomic DNA treated with ultrafast BS replicate 2; Mus musculus; Bisulfite-Seq

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.