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Troubleshooting the gene-variant workflow

Why is my workflow encountering memory errors?

The most likely cause is that these genes contain an unusually high amount of variants, causing memory errors during the aggregation steps of the workflow. However, longer genes can also cause a problem, for example:

Gene name Ensembl ID
TNNI3 ENSG00000129991
KIF1A ENSG00000130294
ACTN2 ENSG00000077522
PRKAG2 ENSG00000106617
RYR2 ENSG00000198626
SCN5A ENSG00000183873
CRELD1 ENSG00000163703
BBS10 ENSG00000179941

To run the workflow on such problematic genes, the following memory allocations may be used.

In quick_merge.wdl:

Task to be edited Parameter Default Change to...
split memory 1GB 2GB
cpu 1 1
queue short short
first_round_merge memory 20GB 32GB
cpu 1 2
queue short medium
second_round_merge memory 10 48GB
cpu 1 3
queue short medium

In annotation.wdl:

Task to be edited Parameter Default Change to...
fill_tags_query memory allocation 2GB 5GB
number of cores 1 1
queue short short
annotate memory allocation 1GB 5GB
number of cores 4 4
queue short short
sum_and_annotate memory allocation 5GB 10GB
number of cores 1 1
queue short short

Note: A version of the workflow with these memory allocations is not actively supported.

Questions relating to autosomal hemizygosity

What do the AC_count and NS_variant columns mean?

The AC and NS columns produced by bcftools "fill-tags".

Column Description
AC_variant Allele count in genotypes. This is the number of alleles for the given variant counted from the genotypes listed in the VCF.
NS_variant Number of samples with data. This is the number of samples which have been included for this particular variant, and is equal to the number of samples with any variant at the given chromosomal position.

Why is AC_Hemi_variant > 0 when my gene of interest is not on a sex chromosome?

For autosomal variants, the majority of samples will have diploid genotypes (e.g. 0/1). However, some samples will have haploid (hemizygous-like) calls (e.g. 1) for certain variants. Such haploid calls indicate that the respective sample-genotype identified on one chromosome is located within a deletion identified on the other chromosome for the same sample.

These haploid calls are not produced as part of the aggregation procedure, but are seen in the single-sample gVCFs.

Worked Example

In the single-sample gVCF, we have identified the following variant where the genotype is represented as haploid ALT call:

chr1 2118756 A T 1 Haploid ALT genotype identified

On closer inspection in the single-sample gVCF, we see that there is a heterozygous call (0/1) for a 2 bp deletion (TGA > T) 2 bp upstream of the variant (from bases 2118755 - 2118756). Therefore, the A > T SNP above is represented as haploid, because it is located within a known deletion on the other chromosome.

Please note that reference calls spanning that deletion are also haploid (the G reference call).

chr1 2118754 TGA T 0/1 2bp deletion of bases GA from position 2118755 - 2118756. Called as heterozygous (diploid).
chr1 2118755 G . 0 We know the G base in position 2118755 is deleted on one chromosome, but on the other it is REF - therefore the hemizygous genotype 0 is called (haploid).
chr1 2118756 A T 1 We know the A in position 2118756 is deleted on one chromosome, but on the other it is ALT - therefore a hemizygous genotype 1 is called (haploid).