目录

  • 摘要
  • 方法与工具
  • 操作流程
    • 组装
    • 比对
    • 注释
  • 结果展示
    • basemods
    • motif
  • 总结

摘要

前段时间特别忙，一个是项目多，另一个是个人私事，临近月底终于有空可以继续码文章。本篇介绍的是三代甲基化的基本流程分析。在测序时分析序列的甲基化修饰后，使用SMRT官方工具进行分析，得到m4C,m6A,m5C_TET的注释。

方法与工具

测序仪器：Pacbio
分析工具：
组装：Canu；flye
比对：pbmm2；samtools(SMRTlink自带)
注释：ipdSummary,motifmaker(SMRTlink自带)
SMRTlink安装文档
SMRTlink参考文档

操作流程

组装

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bam2fastq转换格式
canu矫正/flye组装
原始数据.bam
原始数据.fastq
组装序列.fasta 
for bam_path in */1.rawdata/*.bam; #读取bam文件路径和样品名
do
sample_id=${bam_path%/1.rawdata/*};
bam2fastq ${sample_id}.bam -o ${sample_id}
canu -correct \-p ${sample_id} -d ../02.Assemble/${sample_id} \genomeSize=6.5m \minReadLength=2000 minOverlapLength=500\corOutCoverage=300 corMinCoverage=2 \-pacbio ${sample_id}.fastq.gzflye --plasmids --pacbio-corr ${sample_id}/${sample_id}.correctedReads.fasta.gz -g 6.5m -o flye/${sample_id}_flye -t 16
done

Canu 自身也可以组装，在这里我们只用了他的校正功能，使用flye进行组装（前同事的pipeline被保留）。当然这两个工具的对比我没有去深究，如果大家有更好的流程可以留言讨论。

比对

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text{fill:#9370db;stroke:none;font-size:10px;font-family:'trebuchet ms', verdana, arial;font-family:var(--mermaid-font-family)}#mermaid-svg-ieRTikiYrAntdRKS g.stateGroup text{fill:#9370db;fill:#333;stroke:none;font-size:10px}#mermaid-svg-ieRTikiYrAntdRKS g.statediagram-cluster .cluster-label text{fill:#333}#mermaid-svg-ieRTikiYrAntdRKS g.stateGroup .state-title{font-weight:bolder;fill:#000}#mermaid-svg-ieRTikiYrAntdRKS g.stateGroup rect{fill:#ECECFF;stroke:#9370db}#mermaid-svg-ieRTikiYrAntdRKS g.stateGroup line{stroke:#9370db;stroke-width:1}#mermaid-svg-ieRTikiYrAntdRKS .transition{stroke:#9370db;stroke-width:1;fill:none}#mermaid-svg-ieRTikiYrAntdRKS .stateGroup .composit{fill:white;border-bottom:1px}#mermaid-svg-ieRTikiYrAntdRKS .stateGroup .alt-composit{fill:#e0e0e0;border-bottom:1px}#mermaid-svg-ieRTikiYrAntdRKS .state-note{stroke:#aa3;fill:#fff5ad}#mermaid-svg-ieRTikiYrAntdRKS .state-note text{fill:black;stroke:none;font-size:10px}#mermaid-svg-ieRTikiYrAntdRKS .stateLabel 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.edge-pattern-dashed{stroke-dasharray:3}#mermaid-svg-ieRTikiYrAntdRKS .edge-pattern-dotted{stroke-dasharray:2}#mermaid-svg-ieRTikiYrAntdRKS .marker{fill:#333}#mermaid-svg-ieRTikiYrAntdRKS .marker.cross{stroke:#333}:root { --mermaid-font-family: "trebuchet ms", verdana, arial;}#mermaid-svg-ieRTikiYrAntdRKS {color: rgba(0, 0, 0, 0.75);font: ;}

pbmm2建立索引.mmi -- pbmm2比对
samtools sort排列
组装序列.fasta
数据比对
原始数据.bam
临时文件.tmp.bam
比对文件.bam 
for bam_path in */1.rawdata/*.bam; #读取bam文件路径和样品名
do
sample_id=${bam_path%/1.rawdata/*};
echo ${bam_path} ${sample_id};
mkdir ../02.Assemble/ref ../05.align#建立参考和比对文件夹
pbmm2 index ../02.Assemble/flye/${sample_id}_assembly.fasta ../02.Assemble/ref/${sample_id}_assembly.mmi #建立参考序列索引
pbmm2 align ../02.Assemble/flye/${sample_id}_assembly.fasta ${bam_path} ../05.align/${sample_id}_tmp.bam #比对
samtools sort ../05.align/${sample_id}_tmp.bam  ../05.align/${sample_id} #排列文件
done

注释

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samtools建立索引.fai
samtools建立索引.bai
组装序列.fasta
motif.gff
比对文件.bam
motifs.csv 
for bam_path in */1.rawdata/*.bam; #读取bam文件路径和样品名
do
mkdir -p ../05.align/methylome/${sample_id}
samtools index ../05.align/${sample_id}.bam
samtools faidx ../02.Assemble/flye/${sample_id}_assembly.fasta
ipdSummary ../05.align/${sample_id}.bam --reference ../02.Assemble/flye/${sample_id}_assembly.fasta --gff ../05.align/methylome/${sample_id}/basemods.gff --csv ../05.align/methylome/${sample_id}/basemods.csv --pvalue 0.001 --numWorkers 16 --identify m4C,m6A,m5C_TET
motifMaker find -f ../02.Assemble/flye/${sample_id}_assembly.fasta -g ../05.align/methylome/${sample_id}/basemods.gff -o ../05.align/methylome/${sample_id}/motifs.csv
motifMaker reprocess -f ../02.Assemble/flye/${sample_id}_assembly.fasta -g ../05.align/methylome/${sample_id}/basemods.gff -m ../05.align/methylome/${sample_id}/motifs.csv -o ../05.align/methylome/${sample_id}/motifs.gff

结果展示

可以得到4个结果，gff和csv各2个文件（M.前缀是自己加的，用于区分样品）

basemods

basemods.gff文件提供了m4C,m6A,m5C_TET的注释信息，csv提供了突变位点的信息

motif

motif.gff注释文件和basemods.gff前面都差不多，只有部分位点最后一列注释信息上有些区别，多一个id=BSC；motif.csv提供甲基化片段序列位置信息，如果序列太少，可以在motifmaker可以设置-m 参数调整筛选分数。

总结

三代的甲基化修饰其实并不复杂，工具安装也很方便好用。个人感觉后续是加一些分析，比如做统计，继续绘制一些图片的。介于目前客户需求，暂时没有添加，有兴趣的朋友可以加群一起交流。遇见二维码过期可添加VX：bbplayer2021 ，备注 申请加入生信交流群。

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