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README.md

gnomad_qc

This repo contains the complete set of scripts used to perform sample and variant QC for the gnomAD v2.1 release. We will continue to update and improve upon the code to handle new releases as they grow in size and complexity and as they require increasingly sophisticated QC treatment. The current code therefore represents the most recent iteration of our pipelines and is guaranteed to change over time.

NB: The scripts make reference to gnomAD-related metadata files (not public) and may perform procedures that are not strictly necessary for quality control of all germline datasets. For example, the gnomAD dataset comprises both exomes and genomes, and a substantial portion of the code is written to handle technical differences between those call sets, as well as to perform relevant joint analyses (such as inferring cryptically related individuals across exomes and genomes). These steps may not be relevant for all call sets.

We therefore encourage users to browse through the code and identify modules and functions that will be useful in their own pipelines, and to edit and reconfigure the gnomAD pipeline to suit their particular analysis and QC needs. A more extensive overview and explanation of the gnomAD v2.1 QC process is available at: https://macarthurlab.org/2018/10/17/gnomad-v2-1/ and may help inform users’ design decisions for other pipelines.

Note also that many basic functions and file paths used in the code are imported from a separate repo, gnomad_hail (https://github.com/macarthur-lab/gnomad_hail). The scripts below are run approximately in the order they are listed.

Contents and ordering of scripts

Quality Control

load_data/

  • import_vcf.py — import raw VCF files into Hail, left-align indel representation, and write to MatrixTable format
  • import_resources.py — import reference datasets (e.g., Clinvar annotations, methylation and CpG annotations, ExAC site annotations, truth sets) into Hail and write to MatrixTable or Table format
  • load_coverage.py — import individual-level coverage files, write to MatrixTable and Table formats, compute summary metrics, and export summary data for release

sample_qc/

  • apply_hard_filters.py — filter dataset to bi-allelic, high-call rate, common SNPs; import and annotate relevant metadata; infer chromosomal sex; annotate samples failing hard filtering thresholds; export relevant annotations for ranking samples for removal due to relatedness (see joint_sample_qc.py)
  • generate_hardcalls.py — generate “hardcalls” version of dataset (dropping extraneous GT fields); adjust for sex ploidy; split multi-allelic sites; generate dataset version with non-reference genotypes only
  • exomes_platform_pca.py — transform exome genotype data into missingness data over exome intervals; run PCA on missingness data; cluster and assign generic platform labels
  • joint_sample_qc.py — join exome and genome data; filter joint callset to high-call rate variants and remove rare variants; LD-prune joint callset; evaluate relatedness and drop related samples; perform genotype PCA on unrelated samples; project related samples onto PCs; fit random forest model on PCs for samples with known ancestry labels; apply RF model to assign ancestry to remaining samples; evaluate sample quality metrics on a joint platform-and-population basis and flag outlier samples
  • assign_subpops.py — annotate LD-pruned joint exome and genome data with hard filters and previously inferred platform and population labels; split joint data by relatedness status; filter to samples in continental ancestry group of interest; filter relateds/unrelateds to high-call rate variants; run genotype PCA on unrelateds; project relateds onto subpop PCs; fit random forest model on subpop PCs for samples with known subpopulation labels; apply RF model to assign subpop ancestry to remaining samples
  • finalize_sample_qc.py — combine hard filter, platform inference, relatedness, ancestry inference, subpopulation inference, and TOPMED annotations into unified metadata tables for both exomes and genomes; define release sample set; collapse small population sizes into “other” category
  • create_fam.py — depends on relatedness output (joint_sample_qc.py) and hard filter flags (apply_hard_filters.py); infer all complete trios from kinship coefficients and sex imputation annotations, including duplicates; select representative set of unique trios in call set; evaluate inferred trios by generating random trios and comparing Mendelian violations for random trios against inferred trios; generate final list of inferred trios by filtering out trios with Mendelian violation counts exceeding standard deviation cutoff
  • get_topmed_dups.py — depends on frequency table annotations (annotations/generate_frequency_data.py); create table of high-quality autosomal SNPs shared across gnomAD and TOPMED along with allele count and singleton/doubleton/tripleton annotations (on variants and samples, respectively) for identifying samples present in both gnomAD and TOPMED

variant_qc/

  • variantqc.py — gather variant- and allele-level annotations used as features for training random forests model, impute missing entries; select variants for training examples; train random forests model using specified parameters for depth and number of trees; if specified, test resulting model on pre-selected region; save training data with metadata describing random forest parameters used; apply random forest model to full variant set; annotate results with binned scores (dependent on create_ranked_scores.py) and create variant filter status based on SNP and indel quality cutoffs, inbreeding coefficient thresholds
  • create_ranked_scores.py — create table with rank annotations based on random forest and/or VQSR variant quality scores, with SNPs and indels handled separately, along with optional additional rankings for more specific variant types (e.g., bi-allelic variants, singletons, bi-allelic singletons); bin the variants in the rank tables by all possible rank groupings and add variant annotations from external validation datasets; compute evaluation metrics for each bin for eventual comparison of variant filtering performance across multiple random forest models and VQSR
  • calculate_concordance.py — compute concordance against truth data (NA12878 and synthetic diploid sample) and/or for duplicate samples across exomes and genomes; create a concordance table binned by rank (both absolute and relative) for a given data type (exomes/genomes), truth sample, and filtering model (e.g., VQSR, random forest) for the purposes of evaluating variant filtering models

Making the Release

annotations/

  • generate_qc_annotations.py — compute and collate all variant- and allele-level annotations used for variant QC, including VEP; allele type; variant type; call stats (AC, AN, AF, homozygote count) for high-quality, release, and all samples; histograms of GQ, DP, and AB metric distributions; and trio statistics (Mendelian errors and transmission disequilibrium test results)
  • generate_frequency_data.py — compute frequencies of variants in gnomAD for various sample groupings (e.g., ancestry, sex, and subpopulation) and for downsampled sets; add filtering allele frequency annotations for ancestry sample groupings

variant_qc/

  • correct_fafs.py — temporary fix to address 2.1 bug; filter allele frequency annotations so that populations in which AC=1 have filtering allele frequencies set to 0
  • prepare_data_release.py — combine frequency, filtering alelle frequency, variant QC, VEP, dbSNP, and variant QC metric histogram annotations into unified table; add frequency annotations for each sample subset (e.g., controls, non-neuro, non-cancer, non-TOPMED); select and reformat release annotations for VCF export; create VCF header text; perform sanity check on release annotations
  • make_var_annot_hists.py — aggregate background distributions of specified variant QC metrics into histograms; first-pass run determines minimum and maximum values per metric; second-pass run aggregates QUAL metrics binned by allele frequency and aggregates other metrics over manually set bins/ranges, writes results out to json files (primarily for browser loading purposes)