参考github GWAS教学：https://github.com/MareesAT/GWA_tutorial/
总流程分为4部分
1. QC
2. Population Stratification,
3. Association Analyses
4. Polygenic Risk Score (PRS) analyses.
首先是下载数据集以及相关文件
mkdir GWAS_test
cd GWAS_test
git clone https://github.com/MareesAT/GWA_tutorial.git
unzip 1_QC_GWAS.zip
cd 1_QC_GWAS
1. QC
qc的内容可以参考文献
Marees AT, de Kluiver H, Stringer S, et al. A tutorial on conducting genome-wide association studies: Quality control and statistical analysis. Int J Methods Psychiatr Res. 2018;27(2):e1608. doi:10.1002/mpr.1608
QC一般分为7步
(1) snp 缺失
(2) 性别筛选
(3) MAF 频率筛选
(4) 哈代温伯格平衡测试
(5) 杂合率
(6) 相关性
(7) 人口分层
1.1 查看是否有SNP，individual缺失。
plink --bfile HapMap_3_r3_1 --missing
Rscript --no-save hist_miss.R
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根据SNPs，individuals缺失值设置阈值进行筛选。
# Delete SNPs with missingness >0.2.
plink --bfile HapMap_3_r3_1 --geno 0.2 --make-bed --out HapMap_3_r3_2
# Delete individuals with missingness >0.2.
plink --bfile HapMap_3_r3_2 --mind 0.2 --make-bed --out HapMap_3_r3_3
# Delete SNPs with missingness >0.02.
plink --bfile HapMap_3_r3_3 --geno 0.02 --make-bed --out HapMap_3_r3_4
# Delete individuals with missingness >0.02.
plink --bfile HapMap_3_r3_4 --mind 0.02 --make-bed --out HapMap_3_r3_5
1.2 性别差异检验
根据男性和女性在X染色体上SNPs分布频率进行性别信息检验，女性F值<0.2；男性F值>0.8将不符合的样本标记为“PROBLEM”.
plink --bfile HapMap_3_r3_5 --check-sex
Rscript --no-save gender_check.R
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从结果看，女性样本中有一个F值>0.2.
处理性别异常个体有两个方法：删除或根据F值校正。这里选择删除的方式。
删除
grep "PROBLEM" plink.sexcheck| awk '{print$1,$2}'> sex_discrepancy.txt
# This command generates a list of individuals with the status ìPROBLEMî.
plink --bfile HapMap_3_r3_5 --remove sex_discrepancy.txt --make-bed --out HapMap_3_r3_6
# This command removes the list of individuals with the status ìPROBLEMî.
校正
plink --bfile HapMap_3_r3_5 --impute-sex --make-bed --out HapMap_3_r3_6
# This imputes the sex based on the genotype information into your data set.
1.3 生成仅包含常染色体且去除low MAF的bfile
# Select autosomal SNPs only (i.e., from chromosomes 1 to 22).
awk '{ if ($1 >= 1 && $1 <= 22) print $2 }' HapMap_3_r3_6.bim > snp_1_22.txt
plink --bfile HapMap_3_r3_6 --extract snp_1_22.txt --make-bed --out HapMap_3_r3_7
# Generate a plot of the MAF distribution.
plink --bfile HapMap_3_r3_7 --freq --out MAF_check
Rscript --no-save MAF_check.R
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# Remove SNPs with a low MAF frequency.
plink --bfile HapMap_3_r3_7 --maf 0.05 --make-bed --out HapMap_3_r3_8
# 1073226 SNPs are left
# A conventional MAF threshold for a regular GWAS is between 0.01 or 0.05, depending on sample size.
1.4 去除不符合哈代温伯格平衡（HWE）的SNPs
# Check the distribution of HWE p-values of all SNPs.
plink --bfile HapMap_3_r3_8 --hardy
# Selecting SNPs with HWE p-value below 0.00001, required for one of the two plot generated by the next Rscript, allows to zoom in on strongly deviating SNPs.
awk '{ if ($9 <0.00001) print $0 }' plink.hwe>plinkzoomhwe.hwe
Rscript --no-save hwe.R
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# By default the --hwe option in plink only filters for controls.
# Therefore, we use two steps, first we use a stringent HWE threshold for controls, followed by a less stringent threshold for the case data.
plink --bfile HapMap_3_r3_8 --hwe 1e-6 --make-bed --out HapMap_hwe_filter_step1
# The HWE threshold for the cases filters out only SNPs which deviate extremely from HWE.
# This second HWE step only focusses on cases because in the controls all SNPs with a HWE p-value < hwe 1e-6 were already removed
plink --bfile HapMap_hwe_filter_step1 --hwe 1e-10 --hwe-all --make-bed --out HapMap_3_r3_9
# Theoretical background for this step is given in our accompanying article: https://www.ncbi.nlm.nih.gov/pubmed/29484742 .
1.5 计算杂合率
进行杂合率检验，删除不在mean±3SD范围内的样本
# Checks for heterozygosity are performed on a set of SNPs which are not highly correlated.
# Therefore, to generate a list of non-(highly)correlated SNPs, we exclude high inversion regions (inversion.txt [High LD regions]) and prune the SNPs using the command --indep-pairwiseí.
# The parameters ë50 5 0.2í stand respectively for: the window size, the number of SNPs to shift the window at each step, and the multiple correlation coefficient for a SNP being regressed on all other SNPs simultaneously.
plink --bfile HapMap_3_r3_9 --exclude inversion.txt --range --indep-pairwise 50 5 0.2 --out indepSNP
# Note, don't delete the file indepSNP.prune.in, we will use this file in later steps of the tutorial.
plink --bfile HapMap_3_r3_9 --extract indepSNP.prune.in --het --out R_check
# This file contains your pruned data set.
# Plot of the heterozygosity rate distribution
Rscript --no-save check_heterozygosity_rate.R
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# The following code generates a list of individuals who deviate more than 3 standard deviations from the heterozygosity rate mean.
# For data manipulation we recommend using UNIX. However, when performing statistical calculations R might be more convenient, hence the use of the Rscript for this step:
Rscript --no-save heterozygosity_outliers_list.R
# Output of the command above: fail-het-qc.txt .
# When using our example data/the HapMap data this list contains 2 individuals (i.e., two individuals have a heterozygosity rate deviating more than 3 SD's from the mean).
# Adapt this file to make it compatible for PLINK, by removing all quotation marks from the file and selecting only the first two columns.
sed 's/"// g' fail-het-qc.txt | awk '{print$1, $2}'> het_fail_ind.txt
# Remove heterozygosity rate outliers.
plink --bfile HapMap_3_r3_9 --remove het_fail_ind.txt --make-bed --out HapMap_3_r3_10
1.6相关性检验
Assuming a random population sample we are going to exclude all individuals above the pihat threshold of 0.2 in this tutorial.
# Check for relationships between individuals with a pihat > 0.2.
plink --bfile HapMap_3_r3_10 --extract indepSNP.prune.in --genome --min 0.2 --out pihat_min0.2
# The HapMap dataset is known to contain parent-offspring relations.
# The following commands will visualize specifically these parent-offspring relations, using the z values.
awk '{ if ($8 >0.9) print $0 }' pihat_min0.2.genome>zoom_pihat.genome
# Generate a plot to assess the type of relationship.
Rscript --no-save Relatedness.R
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# The generated plots show a considerable amount of related individuals (explentation plot; PO = parent-offspring, UN = unrelated individuals) in the Hapmap data, this is expected since the dataset was constructed as such.
# Normally, family based data should be analyzed using specific family based methods. In this tutorial, for demonstrative purposes, we treat the relatedness as cryptic relatedness in a random population sample.
# In this tutorial, we aim to remove all 'relatedness' from our dataset.
# To demonstrate that the majority of the relatedness was due to parent-offspring we only include founders (individuals without parents in the dataset).
plink --bfile HapMap_3_r3_10 --filter-founders --make-bed --out HapMap_3_r3_11
# Now we will look again for individuals with a pihat >0.2.
plink --bfile HapMap_3_r3_11 --extract indepSNP.prune.in --genome --min 0.2 --out pihat_min0.2_in_founders
# The file 'pihat_min0.2_in_founders.genome' shows that, after exclusion of all non-founders, only 1 individual pair with a pihat greater than 0.2 remains in the HapMap data.
# This is likely to be a full sib or DZ twin pair based on the Z values. Noteworthy, they were not given the same family identity (FID) in the HapMap data.
# For each pair of 'related' individuals with a pihat > 0.2, we recommend to remove the individual with the lowest call rate.
plink --bfile HapMap_3_r3_11 --missing
# Use an UNIX text editor (e.g., vi(m) ) to check which individual has the highest call rate in the 'related pair'.
# Generate a list of FID and IID of the individual(s) with a Pihat above 0.2, to check who had the lower call rate of the pair.
# In our dataset the individual 13291 NA07045 had the lower call rate.
vi 0.2_low_call_rate_pihat.txt
I
13291 NA07045
# Press esc on keyboard!
:x
# Press enter on keyboard
# In case of multiple 'related' pairs, the list generated above can be extended using the same method as for our lone 'related' pair.
# Delete the individuals with the lowest call rate in 'related' pairs with a pihat > 0.2
plink --bfile HapMap_3_r3_11 --remove 0.2_low_call_rate_pihat.txt --make-bed --out HapMap_3_r3_12
################################################################################################################################
# CONGRATULATIONS!! You've just succesfully completed the first tutorial! You are now able to conduct a proper genetic QC.
# For the next tutorial, using the script: 2_Main_script_MDS.txt, you need the following files:
# - The bfile HapMap_3_r3_12 (i.e., HapMap_3_r3_12.fam,HapMap_3_r3_12.bed, and HapMap_3_r3_12.bim
# - indepSNP.prune.in