Methylation calling/extraction

Bismark output discriminates between cytosines in CpG, CHG and CHH context

Strand-specific methylation output files (default):

• OT：original top；
• CTOT：complementary to OT
• OB：original bottom；
• CTOB：complementary to OB

OT和CTOT的甲基化调用将提供原始顶部链上的胞嘧啶甲基化位置的信息，OB和CTOB的调用将提供初始底部链上的甲基化位置信息。

由于胞嘧啶可以存在于三种不同的序列上下文（CpG、CHG或CHH）中的任何一种中，bismark_methylation_extractor默认输出每个输入文件将产生12个单独输出文件（CpG_OT_…、CpG_CTOT_…、CpG_OB_…等）。

bismark_methylation_extractor  \
    --report \
    --buffer_size 8G \
    --cytosine_report \
    --gzip  \
    --bedGraph  \
    --paired-end \
    --multicore 3  \
    --genome_folder data2/index/ \
    -s mapped/test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.bam \
    --output methylation_extracted 2> logs/bismark/test_MappedOn_NC010473.methylation_extractor.log

bismark_methylation_extractor主要产生以下字符串结尾的文件

• .bedGraph.gz
• .bismark.cov.gz
• .M-bias.txt
• _splitting_report.txt

methylation_extracted
├──[CHG_CTOB_]test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.txt.gz
├──[CHG_CTOT_]test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.txt.gz
├──[ CHG_OB_]test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.txt.gz
├──[ CHG_OT_]test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.txt.gz
├──[ CHH_CTOB_]test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.txt.gz
├──[ CHH_CTOT_]test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.txt.gz
├──[ CHH_OB_test_]MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.txt.gz
├──[ CHH_OT_test_]MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.txt.gz
├──[ CpG_CTOB_test_]MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.txt.gz
├──[ CpG_CTOT_]test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.txt.gz
├──[ CpG_OB_test_]MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.txt.gz
├──[ CpG_OT_test_]MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.txt.gz
├── test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted[.bedGraph.gz]
├── test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted[.bismark.cov.gz]
├── test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted[.M-bias.txt]
└── test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted[_splitting_report.txt]

Context-dependent methylation output files (--comprehensive option):

如果对链特异性甲基化不感兴趣，所有可用的甲基化信息都可以合并到一个上下文相关的文件中（来自四条链中任何一条的信息都将合并）。这将默认为三个输出文件（CpG-context and Non-CpG-context），如果选择了--merge_Non_CpG则会生成两个输出文件（请注意，这可能会导致非CpG输出的文件大小过大）。

bismark_methylation_extractor  \
    --comprehensive \
    --report \
    --buffer_size 8G \
    --cytosine_report \
    --gzip  \
    --bedGraph  \
    --paired-end \
    --multicore 3  \
    --genome_folder data2/index/ \
    -s mapped/test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.bam \
    --output methylation_extracted 2> logs/bismark/test_MappedOn_NC010473.methylation_extractor.log

methylation_extracted
├── CHG_context_test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.txt.gz
├── CHH_context_test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.txt.gz
├── CpG_context_test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.txt.gz
├── test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.bedGraph.gz
├── test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.bismark.cov.gz
├── test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.M-bias.txt
└── test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted_splitting_report.txt

Cytosine Methylation after Extraction

test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted_splitting_report.txt

test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.bam

Parameters used to extract methylation information:
Bismark Extractor Version: v0.24.0
Bismark result file: paired-end (SAM format)
Ignoring first 2 bp of Read 2
Output specified: strand-specific (default)
No overlapping methylation calls specified


Processed 461 lines in total
Total number of methylation call strings processed: 922

Final Cytosine Methylation Report
=================================
Total number of C's analysed:   16852

Total methylated C's in CpG context:    340
Total methylated C's in CHG context:    240
Total methylated C's in CHH context:    988

Total C to T conversions in CpG context:        192
Total C to T conversions in CHG context:        2974
Total C to T conversions in CHH context:        12118

C methylated in CpG context:    63.9%
C methylated in CHG context:    7.5%
C methylated in CHH context:    7.5%

M-Bias Plot

test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.M-bias.txt

其它输出文件

CHG_context_test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.txt.gz

• z - C in CpG context - unmethylated
• Z - C in CpG context - methylated
• x - C in CHG context - unmethylated
• X - C in CHG context - methylated
• h - C in CHH context - unmethylated
• H - C in CHH context - methylated
• u - C in Unknown context (CN or CHN) - unmethylated
• U - C in Unknown context (CN or CHN) - methylated
• . - not a C or irrelevant position

test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.bedGraph.gz

test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.bismark.cov.gz

甲基化水平计算

甲基化水平可根据未转化为 T 的 C 与转化为 T 的 C 的 reads 的比例计算得到，即：
Beta-value = C-reads / (C-reads + T-reads) * 100%
其中，Beta-value 即为该胞嘧啶的甲基化水平，C-reads 为覆盖该位点的支持甲基化的reads 数目（测得该位点为 C 的 reads），T-reads 为覆盖该位点的不支持甲基化的 reads 数目（测得该位点为 T 的 reads）。计算原理示意图如下：

bismark_methylation_extractor结果解释

Optional bedGraph output

bismark2bedGraph  \
    --CX methylation_extracted/CpG_context_test_MappedOn_NC010473_trim_bismark_pe.deduplicated.sorted.txt.gz \
    -o test_MappedOn_NC010473_CpG --dir coverage 

coverage
├── test_MappedOn_NC010473_CpG
└── test_MappedOn_NC010473_CpG.gz.bismark.cov

test_MappedOn_NC010473_CpG

test_MappedOn_NC010473_CpG.gz.bismark.cov

Optional genome-wide cytosine report output

Bismark甲基化提取器还可以输出全基因组胞嘧啶甲基化报告。它也按染色体坐标排序，但也包含序列上下文

coverage2cytosine  --CX \
    --genome_folder data2/index/ \
    -o coverage/test_MappedOn_NC010473_CpG \
    --dir . coverage/test_MappedOn_NC010473_CpG