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NGS-PCA

Methods for running PCA on NGS data

Install mosdepth

Install mosdepth using the instructions from https://github.com/brentp/mosdepth#installation. There are lots of ways to do this, including downloading a linux executable, as a Docker image, or using brew.

Run mosdepth on bams or crams

The "--by 1000" (compute coverage on 1000bp bins) is really the only important argument, and each run is going to look something like this: mosdepth -n -t 1 --by 1000 --fasta /path/to/GRCh38_full_analysis_set_plus_decoy_hla.fa output_filename input_filename.bam

but here is an example script to iterate over all BAM files in a directory (could be run on CRAM files as well)

ref=/path/to/GRCh38_full_analysis_set_plus_decoy_hla.fa

dirOfBams=/path/to/bams/
mosdepthResultsDir=/path/to/mosdepthOutput/
mosdepthThreads=1
parallelThreads=24

find "$dirOfBams" -type f -name "*.bam" \
|parallel -j $parallelThreads "mosdepth -n -t $mosdepthThreads --by 1000 --fasta $ref $mosdepthResultsDir{/.}.by1000 {}"

Run ngs pca

The number of PCs to compute should be in the range of 5% of your sample size and likely far more than you'll actually use. We're still working on optimizing the number of iterations and oversample parameter - but this should be reasonable. For smaller sample sizes it may be worth testing a range of -iters arguments (10,20,30,40,50,100,etc). More iterations increases the accuracy of the PCs, but also increases compute time. For larger sample sizes (10K+), 10 iterations appears to be sufficient.

This will generate svd.pcs.txt in the output directory

ngsPCAOutputDir=/path/to/ngsPCA/
ngsPCAThreads=24
# number of PCs to compute, will likely only use ~10 for 700 samples, so computing 100 should be plenty to play with
numPCs=100

ngsPCAExcludeRegions=ngs_pca_exclude.sv_blacklist.map.kmer.50.1.0.dgv.gsd.sorted.merge.bed
jar=$HOME/ngspca.jar

java -Xmx60G -jar "$jar" \
-input $mosDepthResultsDir \
-outputDir $ngsPCAOutputDir \
-numPC $numPCs \
-sampleEvery 0 \
-threads $ngsPCAThreads \
-iters 	40 \
-randomSeed 42 \
-oversample 100 \
-bedExclude $ngsPCAExcludeRegions

Exclude bed

ngs_pca_exclude.sv_blacklist.map.kmer.50.1.0.dgv.gsd.sorted.merge.bed can be found here. This bed file is suitable for analysis of GRCh38/hg38 WGS samples.

For GRCh38/hg38 WES analysis, the WGS exclude bed file can be concatenated with the bed file that defines the exome targets, where the targets have first been buffered by 20kb. A pre-made WES exclude bed suitable for UKB samples can be found here. The original targets used to generate this file are sourced from http://biobank.ndph.ox.ac.uk/showcase/refer.cgi?id=3801 and can be retrieved with wget -nd biobank.ndph.ox.ac.uk/showcase/showcase/auxdata/xgen_plus_spikein.b38.bed

Brief pipeline description

The jar can be downloaded from a release https://github.com/PankratzLab/NGS-PCA/releases or be run from docker

The ngspca jar will essentially:

  1. Select autosomal bins that do not overlap any region in the excluded bed
  2. Normalize input data
    • Normalize within sample by computing fold change
      • log2(coverage of bin / median coverage of all selected bins)
    • Center each bin to median fold-change of 0 across all samples
  3. Perform Randomized PCA