Allele Mismatches & Strand Flips

[alkaZeltser]

Allele Matching

The first step in applying a PGS to genetic data is to match up the variant records between the PGS weight data and the genetic data (such as a VCF file). ApplyPolygenicScore does this by matching variant coordinates (chromosome and base pair). A common subsequent quality check is to verify whether the alleles associated with each variant also match between the two datasets. By convention, the expectation is that the effect allele from the PGS weight data should match the alternative (ALT) allele in the VCF record, and the other (non-effect) PGS weight allele should match the reference (REF) allele in the VCF record. For example, the ideal case is shown here:

other_allele (PGS)	effect_allele (PGS)	REF allele (VCF)	ALT allele (VCF)
A	C	A	C

Most of the time, these allele pairs will match up, but there are scenarios where that is not the case. A possible consequence of a mismatched set of alleles is an incorrect computation of the effect allele dosage, resulting in an incorrect final PGS sum.

Effect switch

The most common cause of an other/REF & effect/ALT allele mismatch is when the labeling of the effect allele breaks convention, assigning the effect weight to the REF allele instead.

other_allele (PGS)	effect_allele (PGS)	REF allele (VCF)	ALT allele (VCF)
A	C	C	A

The apply.polygenic.score function in our package does not assume that only the ALT allele can be an effect allele. A "risk" dosage for each variant is computed relative to the effect_allele as labeled in the PGS weight data, and no special action is required to correct for a potential effect label switch.

Strand flip

Another scenario results from a strand flip. "Strand" refers to the fact that the DNA molecule structure is composed of two strands. The base sequences along the two strands are each other's reverse complement. Bases on one strand are always paired with the same base on the reverse strand. These are the pairings:

Forward Strand Base	Reverse Strand Base
A	T
T	A
C	G
G	C

When variant calling or genotyping is performed, variants are called relative to a reference DNA sequence, and a decision must be made as to which of the two strands should be used as the reference. By convention, most modern studies call variants relative to the forward strand. However, early genotyping arrays would sometimes include probes for the reverse strand, often because of superior performance. If the PGS was developed on data that included a “flipped” variant site, but the VCF data is consistently called against the forward strand, the result is an allele mismatch:

other_allele (PGS)	effect_allele (PGS)	REF allele (VCF)	ALT allele (VCF)
A	C	T	G

This scenario is simple to check for and correct. If an allele mismatch is detected, all that is necessary is to flip one set of alleles to their opposites, and verify the match once more. If the flipped alleles now match between the two datasets, the strand flip has been resolved. ApplyPolygenicScore provides the assess.pgs.vcf.allele.match function which will perform a strand check and correctional flip for any two pairs of alleles. This same function is used internally by apply.polygenic.score to optionally perform the same action after the PGS/VCF merge step and before the dosage computation step.

There are several caveats to this strategy.

• Both the other_allele and effect_allele in the PGS weight data are required for a strand flip check. Unfortunately, the other allele is not always reported in PGS weight files, and is not a requirement of PGS Catalog submisisons.
• Some strand flips are impossible to distinguish from effect allele switches, resulting in an ambiguous allele match. This happens in palindromic allele scenarios, when the two alleles are also each other's reverse complements:

other_allele (PGS)	effect_allele (PGS)	REF allele (VCF)	ALT allele (VCF)
A	T	T	A

• The simple flip check is extremely difficult to implement with INDEL variants (not currently supported by assess.pgs.vcf.allele.match). This is because VCF convention reports INDEL alleles relative to a downstream base. Since the downstream direction will differ by DNA strand, it is impossible to recover some of the INDEL allele sequence for a proper comparison.

assess.pgs.vcf.allele.match and apply.polygenic.score cannot resolve these scenarios, but they do provide options for what to do with an ambiguous allele match: leave as is, or remove the variant from PGS application.