plink进行阈性状（质量性状）关联分析有两种方法
--assoc 和 --logistic，
这两种方法又分别有分是否校正：
--assoc --adjust 和 --logistic --adjust
--logistic方法有可以选择是否添加协变量：
--logistic --covar 和 --logistic --adjust --covar
这么算的话一共有6种组合。
1、测试数据
root@PC1:/home/test3# ls qc3.map qc3.ped root@PC1:/home/test3# wc -l * ## 288样本， 417105位点 417105 qc3.map 288 qc3.ped 426702 total root@PC1:/home/test3# head -n 5 qc3.map 1 1:3174 0 3174 1 1:32399 0 32399 1 1:32402 0 32402 1 1:32406 0 32406 1 1:32443 0 32443 root@PC1:/home/test3# cut -f 1-10 qc3.ped | head -n 5 ## ped文件的第5列性别，第6列是表型数据，1或者2 1 1 0 0 1 1 T C T C 2 2 0 0 1 1 C C C C 3 3 0 0 1 1 C C C C 4 4 0 0 1 2 C C C C 5 5 0 0 1 1 C C C C
2、 --assoc
root@PC1:/home/test3# ls qc3.map qc3.ped root@PC1:/home/test3# plink --file qc3 --assoc --out test ## 直接使用assoc命令 PLINK v1.90b6.24 64-bit (6 Jun 2021) www.cog-genomics.org/plink/1.9/ (C) 2005-2021 Shaun Purcell, Christopher Chang GNU General Public License v3 Logging to test.log. Options in effect: --assoc --file qc3 --out test 15974 MB RAM detected; reserving 7987 MB for main workspace. .ped scan complete (for binary autoconversion). Performing single-pass .bed write (417105 variants, 288 people). --file: test-temporary.bed + test-temporary.bim + test-temporary.fam written. 417105 variants loaded from .bim file. 288 people (288 males, 0 females) loaded from .fam. 288 phenotype values loaded from .fam. Using 1 thread (no multithreaded calculations invoked). Before main variant filters, 288 founders and 0 nonfounders present. Calculating allele frequencies... done. Total genotyping rate is 0.948564. 417105 variants and 288 people pass filters and QC. Among remaining phenotypes, 75 are cases and 213 are controls. Writing C/C --assoc report to test.assoc ... done. root@PC1:/home/test3# ls qc3.map qc3.ped test.assoc test.log root@PC1:/home/test3# wc -l * 417105 qc3.map 288 qc3.ped 417106 test.assoc 27 test.log 834526 total root@PC1:/home/test3# head -n 3 test.assoc ## 查看结果 CHR SNP BP A1 F_A F_U A2 CHISQ P OR 1 1:3174 3174 T 0.1351 0.1332 C 0.003612 0.9521 1.017 1 1:32399 32399 T 0.1324 0.1288 C 0.0114 0.915 1.032
3、--assoc --adjust命令
root@PC1:/home/test3# ls qc3.map qc3.ped root@PC1:/home/test3# plink --file qc3 --assoc --adjust --out test ## 直接使用--assoc --adjust PLINK v1.90b6.24 64-bit (6 Jun 2021) www.cog-genomics.org/plink/1.9/ (C) 2005-2021 Shaun Purcell, Christopher Chang GNU General Public License v3 Logging to test.log. Options in effect: --adjust --assoc --file qc3 --out test 15974 MB RAM detected; reserving 7987 MB for main workspace. .ped scan complete (for binary autoconversion). Performing single-pass .bed write (417105 variants, 288 people). --file: test-temporary.bed + test-temporary.bim + test-temporary.fam written. 417105 variants loaded from .bim file. 288 people (288 males, 0 females) loaded from .fam. 288 phenotype values loaded from .fam. Using 1 thread (no multithreaded calculations invoked). Before main variant filters, 288 founders and 0 nonfounders present. Calculating allele frequencies... done. Total genotyping rate is 0.948564. 417105 variants and 288 people pass filters and QC. Among remaining phenotypes, 75 are cases and 213 are controls. Writing C/C --assoc report to test.assoc ... done. --adjust: Genomic inflation est. lambda (based on median chisq) = 1.18741. --adjust values (417105 variants) written to test.assoc.adjusted . root@PC1:/home/test3# ls qc3.map qc3.ped test.assoc test.assoc.adjusted test.log root@PC1:/home/test3# wc -l * 417105 qc3.map 288 qc3.ped 417106 test.assoc 417106 test.assoc.adjusted 30 test.log 1251635 total root@PC1:/home/test3# head -n 3 test.assoc ## 查看结果 CHR SNP BP A1 F_A F_U A2 CHISQ P OR 1 1:3174 3174 T 0.1351 0.1332 C 0.003612 0.9521 1.017 1 1:32399 32399 T 0.1324 0.1288 C 0.0114 0.915 1.032 root@PC1:/home/test3# head -n 3 test.assoc.adjusted ## 查看结果 CHR SNP UNADJ GC BONF HOLM SIDAK_SS SIDAK_SD FDR_BH FDR_BY 20 20:11254823 7.14e-48 1.357e-40 2.978e-42 2.978e-42 INF INF 1.489e-42 2.013e-41 20 20:11269211 7.14e-48 1.357e-40 2.978e-42 2.978e-42 INF INF 1.489e-42 2.013e-41
4、--logistic
root@PC1:/home/test3# ls qc3.map qc3.ped root@PC1:/home/test3# plink --file qc3 --logistic --out test ## 直接使用logistic PLINK v1.90b6.24 64-bit (6 Jun 2021) www.cog-genomics.org/plink/1.9/ (C) 2005-2021 Shaun Purcell, Christopher Chang GNU General Public License v3 Logging to test.log. Options in effect: --file qc3 --logistic --out test 15974 MB RAM detected; reserving 7987 MB for main workspace. .ped scan complete (for binary autoconversion). Performing single-pass .bed write (417105 variants, 288 people). --file: test-temporary.bed + test-temporary.bim + test-temporary.fam written. 417105 variants loaded from .bim file. 288 people (288 males, 0 females) loaded from .fam. 288 phenotype values loaded from .fam. Using 1 thread (no multithreaded calculations invoked). Before main variant filters, 288 founders and 0 nonfounders present. Calculating allele frequencies... done. Total genotyping rate is 0.948564. 417105 variants and 288 people pass filters and QC. Among remaining phenotypes, 75 are cases and 213 are controls. Writing logistic model association results to test.assoc.logistic ... done. root@PC1:/home/test3# ls qc3.map qc3.ped test.assoc.logistic test.log root@PC1:/home/test3# wc -l * 417105 qc3.map 288 qc3.ped 417106 test.assoc.logistic 27 test.log 834526 total root@PC1:/home/test3# head test.assoc.logistic ## 查看结果 CHR SNP BP A1 TEST NMISS OR STAT P 1 1:3174 3174 T ADD 273 1.019 0.06304 0.9497 1 1:32399 32399 T ADD 266 1.029 0.1025 0.9183 1 1:32402 32402 C ADD 267 0.9198 -0.4321 0.6657 1 1:32406 32406 C ADD 267 0.8815 -0.6626 0.5076 1 1:32443 32443 C ADD 268 1.16 0.6713 0.502 1 1:32548 32548 T ADD 269 1.038 0.147 0.8831 1 1:45044 45044 A ADD 286 0.8389 -0.7204 0.4713 1 1:45047 45047 T ADD 283 0.8288 -0.6577 0.5107 1 1:45085 45085 C ADD 283 1.007 0.03192 0.9745
5、--logistic --adjust命令
root@PC1:/home/test3# ls qc3.map qc3.ped root@PC1:/home/test3# root@PC1:/home/test3# root@PC1:/home/test3# ls qc3.map qc3.ped root@PC1:/home/test3# plink --file qc3 --logistic --adjust --out test ## 直接运行 PLINK v1.90b6.24 64-bit (6 Jun 2021) www.cog-genomics.org/plink/1.9/ (C) 2005-2021 Shaun Purcell, Christopher Chang GNU General Public License v3 Logging to test.log. Options in effect: --adjust --file qc3 --logistic --out test 15974 MB RAM detected; reserving 7987 MB for main workspace. .ped scan complete (for binary autoconversion). Performing single-pass .bed write (417105 variants, 288 people). --file: test-temporary.bed + test-temporary.bim + test-temporary.fam written. 417105 variants loaded from .bim file. 288 people (288 males, 0 females) loaded from .fam. 288 phenotype values loaded from .fam. Using 1 thread (no multithreaded calculations invoked). Before main variant filters, 288 founders and 0 nonfounders present. Calculating allele frequencies... done. Total genotyping rate is 0.948564. 417105 variants and 288 people pass filters and QC. Among remaining phenotypes, 75 are cases and 213 are controls. Writing logistic model association results to test.assoc.logistic ... done. --adjust: Genomic inflation est. lambda (based on median chisq) = 1.12802. --adjust values (417105 variants) written to test.assoc.logistic.adjusted . root@PC1:/home/test3# ls ## 查看生成的结果 qc3.map qc3.ped test.assoc.logistic test.assoc.logistic.adjusted test.log root@PC1:/home/test3# wc -l * 417105 qc3.map 288 qc3.ped 417106 test.assoc.logistic 417106 test.assoc.logistic.adjusted 30 test.log 1251635 total root@PC1:/home/test3# head -n 3 test.assoc.logistic ## 查看结果 CHR SNP BP A1 TEST NMISS OR STAT P 1 1:3174 3174 T ADD 273 1.019 0.06304 0.9497 1 1:32399 32399 T ADD 266 1.029 0.1025 0.9183 root@PC1:/home/test3# head -n 3 test.assoc.logistic.adjusted ## 查看结果 CHR SNP UNADJ GC BONF HOLM SIDAK_SS SIDAK_SD FDR_BH FDR_BY 20 20:11020547 1.411e-23 4.399e-21 5.887e-18 5.887e-18 INF INF 5.887e-18 7.959e-17 20 20:11403719 4.34e-22 9.205e-20 1.81e-16 1.81e-16 2.22e-16 2.22e-16 9.052e-17 1.224e-15
6、加入协变量， 如何生成协变量
step1：
root@PC1:/home/test3# ls qc3.map qc3.ped root@PC1:/home/test3# plink --file qc3 --genome --out cov ## 直接使用--genome命令 PLINK v1.90b6.24 64-bit (6 Jun 2021) www.cog-genomics.org/plink/1.9/ (C) 2005-2021 Shaun Purcell, Christopher Chang GNU General Public License v3 Logging to cov.log. Options in effect: --file qc3 --genome --out cov 15974 MB RAM detected; reserving 7987 MB for main workspace. .ped scan complete (for binary autoconversion). Performing single-pass .bed write (417105 variants, 288 people). --file: cov-temporary.bed + cov-temporary.bim + cov-temporary.fam written. 417105 variants loaded from .bim file. 288 people (288 males, 0 females) loaded from .fam. 288 phenotype values loaded from .fam. Using up to 8 threads (change this with --threads). Before main variant filters, 288 founders and 0 nonfounders present. Calculating allele frequencies... done. Total genotyping rate is 0.948564. 417105 variants and 288 people pass filters and QC. Among remaining phenotypes, 75 are cases and 213 are controls. IBD calculations complete. Finished writing cov.genome . root@PC1:/home/test3# ls cov.genome cov.log qc3.map qc3.ped
step2：
root@PC1:/home/test3# ls cov.genome cov.log qc3.map qc3.ped root@PC1:/home/test3# plink --file qc3 --read-genome cov.genome --cluster --mds-plot 10 --out cov2 PLINK v1.90b6.24 64-bit (6 Jun 2021) www.cog-genomics.org/plink/1.9/ (C) 2005-2021 Shaun Purcell, Christopher Chang GNU General Public License v3 Logging to cov2.log. Options in effect: --cluster --file qc3 --mds-plot 10 --out cov2 --read-genome cov.genome 15974 MB RAM detected; reserving 7987 MB for main workspace. .ped scan complete (for binary autoconversion). Performing single-pass .bed write (417105 variants, 288 people). --file: cov2-temporary.bed + cov2-temporary.bim + cov2-temporary.fam written. 417105 variants loaded from .bim file. 288 people (288 males, 0 females) loaded from .fam. 288 phenotype values loaded from .fam. Using 1 thread (no multithreaded calculations invoked). Before main variant filters, 288 founders and 0 nonfounders present. Calculating allele frequencies... done. Total genotyping rate is 0.948564. 417105 variants and 288 people pass filters and QC. Among remaining phenotypes, 75 are cases and 213 are controls. Clustering... done. Cluster solution written to cov2.cluster1 , cov2.cluster2 , and cov2.cluster3 . Performing multidimensional scaling analysis (SVD algorithm, 10 dimensions)... done. MDS solution written to cov2.mds . root@PC1:/home/test3# ls ## 查看结果 cov2.mds为下一步要使用的结果 cov2.cluster1 cov2.cluster2 cov2.cluster3 cov2.log cov2.mds cov.genome cov.log qc3.map qc3.ped
step3：
root@PC1:/home/test3# ls cov2.cluster1 cov2.cluster2 cov2.cluster3 cov2.log cov2.mds cov.genome cov.log qc3.map qc3.ped root@PC1:/home/test3# awk '{print$1, $2, $4, $5, $6, $7, $8, $9, $10, $11, $12, $13}' cov2.mds > cov3.txt ## 整理格式 root@PC1:/home/test3# ls cov2.cluster1 cov2.cluster2 cov2.cluster3 cov2.log cov2.mds cov3.txt cov.genome cov.log qc3.map qc3.ped root@PC1:/home/test3# wc -l cov3.txt 289 cov3.txt root@PC1:/home/test3# head -n 3 cov3.txt ## 查看格式 FID IID C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 1 1 0.0111045 -0.0260147 0.0157171 -0.0128374 -0.00871872 0.015319 -0.0206926 0.0229361 -0.0262635 0.0180982 2 2 -0.00213807 0.0639865 -0.0298421 -0.0296622 0.0138335 0.057727 -0.0341887 -0.0108086 0.0270301 0.0114068
8、--logistic --covar
root@PC1:/home/test3# ls cov3.txt qc3.map qc3.ped root@PC1:/home/test3# plink --file qc3 --covar cov3.txt --logistic --hide-covar --out test ##添加协变量 PLINK v1.90b6.24 64-bit (6 Jun 2021) www.cog-genomics.org/plink/1.9/ (C) 2005-2021 Shaun Purcell, Christopher Chang GNU General Public License v3 Logging to test.log. Options in effect: --covar cov3.txt --file qc3 --hide-covar --logistic --out test Note: --hide-covar flag deprecated. Use e.g. "--linear hide-covar". 15974 MB RAM detected; reserving 7987 MB for main workspace. .ped scan complete (for binary autoconversion). Performing single-pass .bed write (417105 variants, 288 people). --file: test-temporary.bed + test-temporary.bim + test-temporary.fam written. 417105 variants loaded from .bim file. 288 people (288 males, 0 females) loaded from .fam. 288 phenotype values loaded from .fam. Using 1 thread (no multithreaded calculations invoked). --covar: 10 covariates loaded. Before main variant filters, 288 founders and 0 nonfounders present. Calculating allele frequencies... done. Total genotyping rate is 0.948564. 417105 variants and 288 people pass filters and QC. Among remaining phenotypes, 75 are cases and 213 are controls. Writing logistic model association results to test.assoc.logistic ... done. root@PC1:/home/test3# ls ## 查看生成结果 cov3.txt qc3.map qc3.ped test.assoc.logistic test.log root@PC1:/home/test3# wc -l * 289 cov3.txt 417105 qc3.map 288 qc3.ped 417106 test.assoc.logistic 31 test.log 834819 total root@PC1:/home/test3# head test.assoc.logistic -n 3 ## 查看结果 CHR SNP BP A1 TEST NMISS OR STAT P 1 1:3174 3174 T ADD 273 0.9015 -0.3232 0.7465 1 1:32399 32399 T ADD 266 0.9416 -0.1934 0.8467
9、--logistic --adjust --covar
root@PC1:/home/test3# ls cov3.txt qc3.map qc3.ped root@PC1:/home/test3# plink --file qc3 --covar cov3.txt --adjust --logistic --hide-covar --out test PLINK v1.90b6.24 64-bit (6 Jun 2021) www.cog-genomics.org/plink/1.9/ (C) 2005-2021 Shaun Purcell, Christopher Chang GNU General Public License v3 Logging to test.log. Options in effect: --adjust --covar cov3.txt --file qc3 --hide-covar --logistic --out test Note: --hide-covar flag deprecated. Use e.g. "--linear hide-covar". 15974 MB RAM detected; reserving 7987 MB for main workspace. .ped scan complete (for binary autoconversion). Performing single-pass .bed write (417105 variants, 288 people). --file: test-temporary.bed + test-temporary.bim + test-temporary.fam written. 417105 variants loaded from .bim file. 288 people (288 males, 0 females) loaded from .fam. 288 phenotype values loaded from .fam. Using 1 thread (no multithreaded calculations invoked). --covar: 10 covariates loaded. Before main variant filters, 288 founders and 0 nonfounders present. Calculating allele frequencies... done. Total genotyping rate is 0.948564. 417105 variants and 288 people pass filters and QC. Among remaining phenotypes, 75 are cases and 213 are controls. Writing logistic model association results to test.assoc.logistic ... done. --adjust: Genomic inflation est. lambda (based on median chisq) = 1.17784. --adjust values (417105 variants) written to test.assoc.logistic.adjusted . root@PC1:/home/test3# ls ### 查看生成结果 cov3.txt qc3.map qc3.ped test.assoc.logistic test.assoc.logistic.adjusted test.log root@PC1:/home/test3# wc -l * 289 cov3.txt 417105 qc3.map 288 qc3.ped 417106 test.assoc.logistic 417106 test.assoc.logistic.adjusted 34 test.log 1251928 total root@PC1:/home/test3# head test.assoc.logistic -n 3 ## 查看结果 CHR SNP BP A1 TEST NMISS OR STAT P 1 1:3174 3174 T ADD 273 0.9015 -0.3232 0.7465 1 1:32399 32399 T ADD 266 0.9416 -0.1934 0.8467 root@PC1:/home/test3# head test.assoc.logistic.adjusted -n 3 ## 查看结果 CHR SNP UNADJ GC BONF HOLM SIDAK_SS SIDAK_SD FDR_BH FDR_BY 20 20:11403719 7.848e-19 3.194e-16 3.274e-13 3.274e-13 3.274e-13 3.274e-13 3.274e-13 4.425e-12 20 20:11020547 2.313e-18 8.011e-16 9.646e-13 9.646e-13 9.646e-13 9.646e-13 3.76e-13 5.082e-12
参考：
（1）https://github.com/MareesAT/GWA_tutorial/
（2）https://blog.csdn.net/yijiaobani/article/details/105294261