Conpair: 配对样本一致性concordance与污染contamination分析

Conpair 于2016年被发表在《Bioinformatics》上，用于分析配对样本（如某个病人的肿瘤样本和正常样本）WGS或WES测序的一致性和交叉个体污染。

特点

• 支持的基因组

因为需要指定markers选项，作者只提供了GRCh37, GRCh38, GRCm38的文件。

• Python版本

python2与python3均适用，这是作者在0.2版本做出的修改。

• 消耗时间

整体耗时较短，使用数分钟就可以完成Conpair分析

示例

run_gatk_pileup_for_sample.py

• 调用GATK的Pileup功能进行分析，不想太麻烦就用GATK3版本。

Tumor

~/miniconda3/envs/py27/bin/python ~/biosoft/Conpair/scripts/run_gatk_pileup_for_sample.py \
--bam WES_FD_T_1.bam \
--outfile WES_FD_T_1_pileup \
--conpair_dir ~/biosoft/Conpair/ \
--reference ~/db/ref/ucsc-human-hg38/hg38.fa \
--markers ~/biosoft/Conpair/data/markers/GRCh38.autosomes.phase3_shapeit2_mvncall_integrated.20130502.SNV.genotype.sselect_v4_MAF_0.4_LD_0.8.liftover.bed \
--gatk ~/miniconda3/envs/rna/share/gatk4-4.5.0.0-0/gatk-package-4.5.0.0-local.jar \
--java ~/miniconda3/envs/rna/bin/java \
--xmx_java 20g > WES_FD_T_1_gatk_pileup.log 2>&1

Normal

~/miniconda3/envs/py27/bin/python ~/biosoft/Conpair/scripts/run_gatk_pileup_for_sample.py \
--bam WES_FD_N_1.bam \
--outfile WES_FD_N_1_pileup \
--conpair_dir ~/biosoft/Conpair/ \
--reference ~/db/ref/ucsc-human-hg38/hg38.fa \
--markers ~/biosoft/Conpair/data/markers/GRCh38.autosomes.phase3_shapeit2_mvncall_integrated.20130502.SNV.genotype.sselect_v4_MAF_0.4_LD_0.8.liftover.bed \
--gatk ~/miniconda3/envs/rna/bin/gatk \
--java "" \
--xmx_java 20g > WES_FD_N_1_gatk_pileup.log 2>&1

verify_concordance.py

• 作者建议是添加-H,--normal_homozygous_markers_only 选项移除拷贝数变对变异的影响。添加后一致性会增高concordance

~/miniconda3/envs/py27/bin/python ~/biosoft/Conpair/scripts/verify_concordance.py \
-T WES_FD_T_1_pileup \
-N WES_FD_N_1_pileup \
--outfile WES_FD_1_concordance.txt \
--markers ~/biosoft/Conpair/data/markers/GRCh38.autosomes.phase3_shapeit2_mvncall_integrated.20130502.SNV.genotype.sselect_v4_MAF_0.4_LD_0.8.liftover.txt \
--conpair_dir ~/biosoft/Conpair/ \
--min_cov 10 \
--min_mapping_quality 10 \
--min_base_quality 20 > WES_FD_1_verify_concordance.log 2>&1

不加-H选项结果

$ cat WES_FD_1_concordance.txt
Concordance: 71.68%
Based on 6092/7353 markers (coverage per marker threshold : 10 reads)
Minimum mappinq quality: 10
Minimum base quality: 20

加-H选项结果

$ cat WES_FD_1_concordance_H.txt
Concordance: 96.33%
Based on 3431/7353 markers (coverage per marker threshold : 10 reads)
Minimum mappinq quality: 10
Minimum base quality: 20

estimate_tumor_normal_contamination.py

• 肿瘤样本即使污染水平在0.5%也会对体细胞突变calling产生严重影响，从而导致特异性降低。

~/miniconda3/envs/py27/bin/python ~/biosoft/Conpair/scripts/estimate_tumor_normal_contamination.py \
-T WES_FD_T_1_pileup \
-N WES_FD_N_1_pileup \
--outfile WES_FD_1_contamination.txt \
--markers ~/biosoft/Conpair/data/markers/GRCh38.autosomes.phase3_shapeit2_mvncall_integrated.20130502.SNV.genotype.sselect_v4_MAF_0.4_LD_0.8.liftover.txt \
--grid 0.01 \
--min_mapping_quality 10 > WES_FD_1_verify_contamination.log 2>&1

结果

$ cat WES_FD_1_contamination.txt
Normal sample contamination level: 0.291%
Tumor sample contamination level: 0.351%