README.md

Imputing and filtering genotypes

This repository contains scripts for imputing and filtering genotypes with IMPUTE2. Other software tools used include plink for pre-processing, shapeit for pre-phasing and snptest for QC.

Some of these scripts were written for use on an SGE cluster and contain commands to submit jobs to the cluster. These submission scripts are appropriate for the environment they were written in but may need to be adapted to run in other environments. The SGE specific scripts are contained in the ./sge sub-directory. This directory also contains wrappers for the more general scripts in the top level directory.

Note that it is assumed that genotypes are stored in ped files, one file per chromosome. File names are assumed to follow the convention chr{chromosome name}.unphased.ped with corresponding map files in the same directory.

The workflow outlined below only deals with autosomes. Imputation of SNPs on sex chromosomes will require some changes.

Reference panel

The IMPUTE2 website provides several reference datasets for download. This includes all files required for imputation.

Pre-phasing genotypes

Prior to to the actual imputation we use shapeit to phase the study genotypes. This allows phasing of entire chromosomes, rather than the small chunks used for imputation, and allows for faster imputation afterwards.

Strand alignment

The code in this section is available as a script from this repository.

Before the study genotypes can be phased it is important to ensure that alleles in the study as well as the reference panel are reported on the same strand. Here we use shapeit's -check option to generate a problem report for the study genotypes.

for chr in $(seq 1 22)
do 
	shapeit -check -P chr${chr}.unphased --input-ref $REF_PREFIX$chr$REF_SUFFIX.hap.gz $REF_PREFIX$chr$REF_SUFFIX.legend.gz  $REF_PREFIX.sample --output-log chr${chr}.alignments
done

From this we can extract a list of all SNPs that need to have their strand flipped or are absent from the reference panel and need to be removed.

for chr in $(seq 1 22)
do
	cat chr$chr.alignments.snp.strand | grep "strand" | cut -f 3 | uniq > chr$chr.strand_flip.lst
	cat chr$chr.alignments.snp.strand | grep "missing" | cut -f 3 | uniq > chr$chr.missing.lst
done

Now we can use plink to write ped files with properly aligned strands and SNPs that are missing in the reference panel excluded.

for chr in $(seq 1 22)
do
	plink --noweb --file chr$chr.unphased --flip chr$chr.strand_flip.lst --exclude chr$chr.missing.lst --recode --out chr$chr.unphased.aligned --make-bed
done

Phasing

The (binary) ped files created above can then be used as input for phasing with shapeit. The getHaplotypes script can be used to submit the necessary jobs to the cluster. Assuming that the ped files are located in the current working directory and that phased haplotypes should be written to the phased/ sub-directory, the following command will generate the necessary jobs on the cluster:

getHaplotypes.sh . phased

Further arguments to shapeit can be passed as additional arguments at the end. Note that this makes assumptions about the location of the reference panel files and the variables at the top of the script may have to be changed if a different reference panel should be used.

Imputation

The phased haplotypes obtained above can be used as input to IMPUTE2, which can then impute genotypes from the reference panel with a single iteration. Imputation is carried out for small regions of the genome rather than entire chromosomes. The imputeChunks script can automatically partition the genome into 5MB chunks and generate the corresponding imputation jobs on an SGE cluster (the command used for job submission can be customised on the command-line).

Quality control

Once genotypes have been imputed they have to be screened for low quality imputations so that these can be removed. Here we use snptest to generate quality metrics for all variants. The submit_snptest script will generate useful summaries from imputed genotypes. By default jobs for several chunks can be generated and submitted to an SGE cluster using this script. To run snptest locally instead use --submit "". The submission script expects four arguments that specify a common prefix and common suffix for the files with imputed genotypes, followed by the name of the sample and output file.

The resulting summary files can then be used to identify SNPs that should be excluded. A list of poor quality genotypes can be obtained with the getExclusions script. This provides facilities to specify upper and lower bounds on the values allowed for columns in the summary file. All SNPs with at least one value outside the specified ranges will be written to an exclusion file. This file, together with all imputed genotypes, will then serve as input to filterGeno.pl, which in turn produces a genotype file with all genotypes that were marked for exclusion removed.

Combining results

Once low quality genotypes have been removed the chunks of imputed genotypes can be merged into larger files, e.g. one per chromosome, using [mergeChunks.pl].

Preparing imputed genotypes for use with Matrix-eQTL

To convert the genotyping data into a format that can be used with Matrix-eQTL the gen2me script can be used.