MuSiC the Mutational Significance In Cancer (MuSiC) suite of tools 官网地址 http://gmt.genome.wustl.edu/packages/genome-music/index.html

功能其主要功能

Apply statistical methods to identify significantly mutated genes
Highlight significantly altered pathways
Investigate the proximity of amino acid mutations in the same gene
Search for gene-based or site-based correlations to mutations and relationships between mutations themselves
Correlate mutations to clinical features, using typical correlation measures, and generalized linear models
Cross-reference findings with relevant databases such as Pfam, COSMIC, and OMIM

软件使用的分析思路

1，生成VCF文件 2，将VCF文件转换成maf（Mutation Annotation Format）文件 3，合并maf文件为一个文件 4，MuSiC bmr calc-covg 生成 covg 文件 5，MuSiC bmr calc-bmr 生成 gene_mrs 文件 6，MuSiC smg 生成 significantly mutated genes 7，Music path-scan 生成 sm_pathways

安装

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sudo apt-add-repository "deb http://apt.genome.wustl.edu/ubuntu lucid-genome main"
wget https://apt.genome.wustl.edu/ubuntu/files/genome-institute.asc
sudo apt-key add genome-institute.asc
sudo apt-get update
# fix dependency error Bit::Vector
sudo cpan install Bit::Vector  
sudo apt-get install genome-music

1，生成VCF文件

可以用varscan2，GATK muTect2等

2，将VCF文件转换成maf

参见前期文章提到的oncotator，要注意通过设置 –annotate-manual=‘Tumor_Sample_Barcode:Tumor_sample_name’ 设置癌症的样本名（对应第16列）

3，合并maf文件

# 人懒，这个文件中间有maf的标题栏，没有去掉 ( ° △ °''')︴ cat *.maf ' grep -v "^#" > myMAF.tsv

4，MuSiC bmr calc-covg 生成 covg 文件

count covered bases per-gene for each given tumor-normal pair of BAMs.

–bam-list 一个专门记录样本的文件，需要配对的bam(需要先index)，共三列，sample_name（样本名） normal_bam（正常组织bam文件） tumor_bam（肿瘤组织bam文件），每个配对样本的sample_name名称要和第二部设置的名称一致。格式如下

aaa /path/to/bam/aaa_normal.bam /path/to/bam/aaa_tumor.bam bbb /path/to/bam/bbb_normal.bam /path/to/bam/bbb_tumor.bam

–roi-file The regions of interest (ROIs) of each gene are typically regions targeted for sequencing or are merged exon loci (from multiple transcripts) of genes with 2-bp flanks (splice junctions).
roi（基于1 base），可以从 https://github.com/ding-lab/calc-roi-covg/blob/master/data/ensembl_67_cds_ncrna_and_splice_sites_hg19 下载到，格式如下，要注意染色体要与maf对应，比如都有chr三个字符。如果maf中的染色体带chr，可以awk ‘{print “chr”$0}’ old.roi > new.chr.roi 。roi文件中的基因名要和maf中对应。

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chr1\t11867\t12229\tDDX11L1
chr1\t12611\t12723\tDDX11L1
chr1\t29552\t30041\tMIR1302-11

如何生成自己需要的roi文件，过几天我可能会讲。

运行命令

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genome music bmr calc-covg 
    --bam-list bam_list  
    --output-dir output_dir/   
    --reference-sequence ref.fa  
    --roi-file new.chr.roi 

文件夹结构

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.
├── gene_covgs
│   ├── aaa.covg
│   └── bbb.covg
├── roi_covgs
│   ├── aaa.covg
│   └── bbb.covg
└── total_covgs
2 directories, 5 files

5，MuSiC bmr calc-bmr 生成 gene_mrs 文件

**Calculates mutation rates given per-gene coverage (from “music bmr calc-covg”), and a mutation list **

由于music不能识别最新的maf中的突变分类中的部分类型，比如lincRNA等（Unrecognized Variant_Classification “lincRNA” in MAF file ），所以要去掉这些列

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sed -i '/lincRNA/d' myMAF.tsv
sed -i '/Stop_Codon_Del/d' myMAF.tsv
sed -i '/Start_Codon_Del/d' myMAF.tsv 
sed -i '/Stop_Codon_Ins/d' myMAF.tsv
sed -i '/Start_Codon_SNP/d' myMAF.tsv
sed -i '/Start_Codon_Ins/d' myMAF.tsv

output-dir 和–roi-file 参数与上步的设置一致

运行

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genome music bmr calc-bmr 
    --bam-list bam_list 
    --maf-file myMAF.tsv  
    --output-dir output_dir/ 
    --reference-sequence ref.fa 
    --roi-file new.chr.roi

6，MuSiC smg 生成 significantly mutated genes

Identify significantly mutated genes

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genome music smg 
      --gene-mr-file output_dir/gene_mrs 
      --output-file output_dir/smgs

结果内容示例如下

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#Gene   Indels  SNVs    Tot Muts        Covd Bps        Muts pMbp       P-value FCPT    P-value LRT     P-value CT      FDR FCPT        FDR LRT FDR CT
GOLGA6L2        4       22      26      7322    3550.94 0       0       0       0       0       0
GPRIN2  2       27      29      2762    10499.64        0       0       0       0       0       0
KCNJ12  0       40      40      2612    15313.94        0       0       0       0       0       0
KRT18   0       18      18      3050    5901.64 3.10862446895044e-15    0       0       4.50964265930054e-12    0       0

7，Music path-scan 生成 sm_pathways

PathScan: a tool for discerning mutational significance in groups of putative cancer genes.

pathway中的KEGG文件可以从https://github.com/ding-lab/parse-kegg/tree/master/all_pathway_files下载运行

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genome music path-scan 
        --bam-list bam_list 
        --gene-covg-dir output_dir/gene_covgs/ 
        --maf-file myMAF.tsv 
        --output-file output_dir/sm_pathways 
        --pathway-file input_dir/pathway_dbs/KEGG.txt    
        --bmr 8.7E-07

sm_pathways的结果内容示例如下

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Pathway Name    Class   Samples_Affected        Total_Variations        p-value FDR
hsa05310        Asthma - Homo sapiens (human)   Human Diseases; Immune System Diseases  2       13      0.88382 1
hsa00062        Fatty acid elongation in mitochondria - Homo sapiens (human)    Metabolism; Lipid Metabolism    2       9       0.92481 0.959873886255924
hsa00130        Ubiquinone and other terpenoid-quinone biosynthesis - Homo sapiens (human)      Metabolism; Metabolism of Cofactors and Vitamins        2       9       0.92689 0.985383058252427

参考：

http://gmt.genome.wustl.edu/packages/genome-music/index.html

http://gmt.genome.wustl.edu/packages/genome-music/install.html

https://www.biostars.org/p/95308/

https://github.com/ding-lab/parse-kegg/tree/master/all_pathway_files

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#Author: Jason

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利用genome music分析癌症样本中显著突变的基因和相关通路

文章目录