Author: Jaehyun Joo

# Processing 1000 Genomes data for set-based association tests

#### Jaehyun Joo

#### 09 September, 2023

## Overview

A set-based association test in the snpsettest package requires a reference data set to infer pairwise linkage disequilibrium (LD) values between a set of variants. This vignette shows you how to use 1000 Genomes data as the reference data for set-based association tests.

## Prerequisites

PLINK 2.0 is required to process the 1000 Genomes dataset. The 1000 Genomes phase 3 dataset (GRCh37) is available in PLINK2 binary format at PLINK 2.0 Resources. To download files,

    # The links in here may be changed in future
    # "-O" to specify output file name
    wget -O all_phase3.psam "https://www.dropbox.com/s/yozrzsdrwqej63q/phase3_corrected.psam?dl=1"
    wget -O all_phase3.pgen.zst "https://www.dropbox.com/s/afvvf1e15gqzsqo/all_phase3.pgen.zst?dl=1"
    wget -O all_phase3.pvar.zst "https://www.dropbox.com/s/op9osq6luy3pjg8/all_phase3.pvar.zst?dl=1"

    # Decompress pgen.zst to pgen
    plink2 --zst-decompress all_phase3.pgen.zst > all_phase3.pgen

## Choose an appropriate population

Patterns of LD could vary among racial/ethnic groups, and thus, it may be necessary to choose an appropriate population. For example, if your GWAS is based on European descent, you may want to keep EUR samples as described in the “all_phase3.psam” file.

    # "vzs" modifier to directly operate with pvar.zst
    # "--chr 1-22" excludes all variants not on the listed chromosomes
    # "--output-chr 26" uses numeric chromosome codes
    # "--max-alleles 2": PLINK 1 binary does not allow multi-allelic variants
    # "--rm-dup" removes duplicate-ID variants
    # "--set-missing-var-id" replaces missing IDs with a pattern
    plink2 --pfile all_phase3 vzs \
           --chr 1-22 \
           --output-chr 26 \
           --max-alleles 2 \
           --rm-dup exclude-mismatch \
           --set-missing-var-ids '@_#_$1_$2' \
           --make-pgen \
           --out all_phase3_autosomes

    # Prepare sub-population filter file
    awk 'NR == 1 || $5 == "EUR" {print $1}' all_phase3.psam > EUR_1kg_samples.txt

    # Generate sub-population fileset
    plink2 --pfile all_phase3_autosomes \
           --keep EUR_1kg_samples.txt \
           --make-pgen \
           --out EUR_phase3_autosomes

## Convert the 1000 Genomes data to PLINK 1 binary format

The snpsettest package uses PLINK 1 binary files to read them into R. The PLINK2 binary fileset (pgen/pvar/psam) can be easily converted to PLINK 1 binary fileset (bed/bim/fam).

    # pgen to bed
    # "--maf 0.005" remove most monomorphic SNPs
    # (still may have some when all samples are heterozyguous -> maf=0.5)
    plink2 --pfile EUR_phase3_autosomes \
           --maf 0.005 \
           --make-bed \
           --out EUR_phase3_autosomes

    # Split bed/bim/fam by chromosome
    for i in {1..22}
    do plink2 --bfile EUR_phase3_autosomes --chr $i --make-bed --out EUR_phase3_chr$i
    done

For the snpsettest package, it is better to split your reference data by chromosome and run set-based association tests with per-chromosome binary files. For instance, when you perform set-based association tests for genes on chromosome 1, you don’t have to load genotype data for other chromosomes into R. Intuitively, as more redundant SNPs are included in the reference data, your tests will get (often significantly) slower and consume more memory.

## Read PLINK 1 binary fileset to R session

This package uses a bed.matrix-class from the gaston package to attach genotype data to R session. Genotypes are retrieved on demand to manage large-scale genotype data.

    library(snpsettest)

    # Read chromosome 1 bed/bim/fam files
    x <- read_reference_bed("/path/to/EUR_phase3_chr1.bed")