Pipeline Overview¶

Our pipeline performs the following steps:

Quality Control¶

Create chunks of 10 Mb each.
For each 10Mb chunk, perform the following checks:

On Chunk Level:
- Determine the number of valid variants: A variant is considered valid if it is included in the reference panel. At least 3 variants must be included.
- Determine the amount of variants found in the reference panel: At least 50% of the variants must be be included in the reference panel.
- Determine the sample call rate: At least 50% of the variants must be called for each sample.
Chunk exclusion:
```
if (#variants < 3 || overlap < 50% || sampleCallRate < 50%) then (exclude chunk)
```
On Variant Level:
- Check alleles: Only A, C, G, T are allowed.
- Calculate alternative allele frequency (AF): Mark all variants with an AF > 0.5.
- Calculate the SNP call rate.
- Calculate the chi-square for each variant (reference panel vs. study data).
- Determine allele switches: Compare the reference and alternate alleles of the reference panel with the study data.
- Determine strand flips: After eliminating possible allele switches, flip and compare the reference and alternate alleles from the reference panel with the study data.
- Determine allele switches in combination with strand flips: Apply the two rules above together.
Variant exclusion: Variants are excluded in case of: [a] invalid alleles occur (!(A,C,G,T)), [b] duplicates (DUP filter or pos - 1 == pos), [c] indels, [d] monomorphic sites, [e] allele mismatch between reference panel and study, [f] SNP call rate < 90%, [g] more than 100 strand or allele switches.

On Sample Level:
- A chunk is excluded if any sample within that chunk has a call rate < 50%. Only complete chunks are excluded, not individual samples (see “On Chunk Level” above).
Perform a liftOver step if the build of the input data does not match the build of the reference panel (e.g., hg37 vs. hg38).

Phasing¶

For each chunk, execute one of the following phasing algorithms (using an overlap of 5 Mb). For example, for chr20:1-20,000,000 and reference population EUR:

Eagle2

./eagle \
   --vcfRef HRC.r1-1.GRCh37.chr20.shapeit3.mac5.aa.genotypes.bcf \
   --vcfTarget chunk_20_0000000001_0020000000.vcf.gz \
   --geneticMapFile genetic_map_chr20_combined_b37.txt \
   --outPrefix chunk_20_0000000001_0020000000.phased \
   --chrom 20 \
   --bpStart 1 \
   --bpEnd 25000000 \
   --allowRefAltSwap \
   --vcfOutFormat z \
   --keepMissingPloidyX \
   --numThreads ${task.cpus}

Target-only sites for unphased data are excluded from the final output.

Imputation¶

For each chunk, execute Minimac4 to impute the phased data (using a window of 500 kb):

    ./Minimac4 \
        --region 20:1-20000000 \
        --overlap 500000 \
        --output ${chunkfile_name}.dose.vcf.gz \
        --output-format vcf.gz \
        --format GT,DS,GP \
        --min-ratio $minimac_min_ratio \
        --all-typed-sites \
        --meta \
        --min-recom 0.00001 \
        --min-r2 0.8 \
        --sites out.info.gz \
        --empirical-output out.empiricalDose.vcf.gz \
        --threads 1 \
        --decay 0 \
        HRC.r1-1.GRCh38.chr1.shapeit3.mac5.aa.genotypes.msav \
        chunk_1_0000000001_0020000000.phased.vcf

Compression and Encryption¶

Merge all chunks of a chromosome into one single VCF file (.vcf.gz).
Encrypt the data with a one-time password.

Chromosome X Pipeline¶

In addition to the standard QC, the following per-sample checks are performed for chrX:

Ploidy Check: Verifies if all variants in the non-PAR region are either haploid or diploid.
Mixed Genotypes Check: Verifies if the proportion of mixed genotypes (e.g., 1/.) is < 10%.

For phasing and imputation, chrX is divided into three independent chunks (PAR1, non-PAR, PAR2). These chunks are then automatically merged by the TOPMed Imputation Server and returned as a single complete chromosome X file.