售后

体细胞突变（somatic mutation）是指患者某些组织或者器官后天性地发生了体细胞变异，虽然它不会遗传给后代个体，却可以通过细胞分裂，遗传给子代细胞。体细胞突变对肿瘤的发生发展有关键性的作用，并且它也是制定肿瘤癌症靶向治疗措施的关键所在。

科学家采取了不同的算法和统计模型用于检测体细胞突变。目前最受欢迎的有Varscan、SomaticSniper、 Strelka 和MuTect2 。这些软件大都是直接对肿瘤-正常样本的每个位点进行比较，对肿瘤样本中明显高于正常样本的次等位基因进行标记，作为体细胞变异，同时排除种系突变和杂合性丢失（LOH）情况。虽然这些软件具有较高的引用率，并在不断地更新，但仍存在不足：

a、缺乏完整可靠的实验来评估检测结果；
b、 缺乏金标准，不能保证检测到的灵敏度和特异性最高；
c、 在实际应用中，各软件的相对优缺点在很大程度上是未知的。

该软件分析需要有正常样本和肿瘤样本，按照有参项目比对得到bam文件，先做如下处理：

排序：

/TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/SNP/bin/java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -jar /TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/SNP/share/gatk4-4.1.1.0-0/gatk-package-4.1.1.0-local.jar ReorderSam -I=/TJPROJ6/RNA_SH/shouhou/202205/MuTect2-test/QC/bam/Control_1.bam -O=/TJPROJ6/RNA_SH/shouhou/202205/MuTect2-test/SNP/Control_1/Control_1_reorder.bam -R=/TJPROJ6/GB_TR/reference_data/new_pip/Animal/Homo_sapiens/Homo_sapiens_Ensemble_94/Homo_sapiens_Ensemble_94.fa --TMP_DIR=/TJPROJ6/RNA_SH/shouhou/202205/MuTect2-test/SNP/Control_1/tmp --VALIDATION_STRINGENCY SILENT

加头：

/TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/SNP/bin/java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -jar /TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/SNP/share/gatk4-4.1.1.0-0/gatk-package-4.1.1.0-local.jar AddOrReplaceReadGroups -I=/TJPROJ6/RNA_SH/shouhou/202205/MuTect2-test/SNP/Control_1/Control_1_reorder.bam -O=/TJPROJ6/RNA_SH/shouhou/202205/MuTect2-test/SNP/Control_1/Control_1_reorder_head.bam -SO=coordinate -LB=LB -PL=illumina -PU=PU -SM=Control --TMP_DIR=/TJPROJ6/RNA_SH/shouhou/202205/MuTect2-test/SNP/Control_1/tmp --VALIDATION_STRINGENCY SILENT

去dup：

/TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/SNP/bin/java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -jar /TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/SNP/share/gatk4-4.1.1.0-0/gatk-package-4.1.1.0-local.jar MarkDuplicates -I=/TJPROJ6/RNA_SH/shouhou/202205/MuTect2-test/SNP/Control_1/Control_1_reorder_head.bam -O=/TJPROJ6/RNA_SH/shouhou/202205/MuTect2-test/SNP/Control_1/Control_1_reorder_head_dedup.bam --VALIDATION_STRINGENCY=SILENT -M=/TJPROJ6/RNA_SH/shouhou/202205/MuTect2-test/SNP/Control_1/Control_1_reorder_head_dedup_metrics.txt -MAX_FILE_HANDLES=4000 --CREATE_INDEX=true --REMOVE_DUPLICATES=true --TMP_DIR=/TJPROJ6/RNA_SH/shouhou/202205/MuTect2-test/SNP/Control_1/tmp

截取reads：

如star比对，比对质量为255为一下命令（gatk4需要转化成hisat2比对质量60）

/TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/SNP/bin/java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -jar /TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/SNP/share/gatk4-4.1.1.0-0/gatk-package-4.1.1.0-local.jar SplitNCigarReads -R=/TJPROJ6/GB_TR/reference_data/new_pip/Animal/Homo_sapiens/Homo_sapiens_Ensemble_94/Homo_sapiens_Ensemble_94.fa -I=/TJPROJ6/RNA_SH/shouhou/202205/MuTect2-test/SNP/Control_1/Control_1_reorder_head_dedup.bam -O=/TJPROJ6/RNA_SH/shouhou/202205/MuTect2-test/SNP/Control_1/Control_1_reorder_head_dedup_split.bam --tmp-dir=/TJPROJ6/RNA_SH/shouhou/202205/MuTect2-test/SNP/Control_1/tmp

如是hisat2比对的bam则需加参数 --skip-mapping-quality-transform，跳过比对质量转化。

Mutect2分析：

/TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/SNP/bin/java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -jar /TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/SNP/share/gatk4-4.1.1.0-0/gatk-package-4.1.1.0-local.jar Mutect2 --reference /TJPROJ6/GB_TR/reference_data/new_pip/Animal/Homo_sapiens/Homo_sapiens_Ensemble_94/Homo_sapiens_Ensemble_94.fa --input /TJPROJ6/RNA_SH/shouhou/202205/MuTect2-test/SNP/Control_1/Control_1_reorder_head_dedup_split.bam --input /TJPROJ6/RNA_SH/shouhou/202205/MuTect2-test/SNP/Treatment_1/Treatment_1_reorder_head_dedup_split.bam --normal-sample Control --output /TJPROJ6/RNA_SH/shouhou/202205/MuTect2-test/bam/muTect2.call.vcf

筛选：

/TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/SNP/bin/java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -jar /TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/SNP/share/gatk4-4.1.1.0-0/gatk-package-4.1.1.0-local.jar FilterMutectCalls --reference /TJPROJ6/GB_TR/reference_data/new_pip/Animal/Homo_sapiens/Homo_sapiens_Ensemble_94/Homo_sapiens_Ensemble_94.fa --variant /TJPROJ6/RNA_SH/shouhou/202205/MuTect2-test/bam/muTect2.call.vcf  --output filter_muTect2.call.vcf

/TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/SNP/bin/java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -jar /TJPROJ2/GB/PUBLIC/software/GB_TR/mRNA/miniconda3/envs/SNP/share/gatk4-4.1.1.0-0/gatk-package-4.1.1.0-local.jar SelectVariants -V=filter_muTect2.call.vcf -O=pass_filter_muTect2.call.vcf --exclude-non-variants  true --exclude-filtered true 

pass_filter_muTect2.call.vcf为最终结果。路径/TJPROJ6/RNA_SH/script_dir/Mutect2-test

重测流程有该软件的使用，有问题可以请教洪翔和韩悦