AggV2 variant normalisation and representation¶

Variants in AggV2 were normalised to decompose multi-allelic variants, make them parsimonious and left-align indels. We used vt (version 0.57721) for this.

Normalisation procedure¶

Normalisation consists of:

Decomposition
Parsimony
Left alignment
Decomposition of bi-allelic block substitutions (MNPs)

Decomposition¶

In the raw VCF output of gVCF genotyper, variants with three or more observed alleles are represented as multi-allelic. We decompose all multi-allelic variants, so that each variant in aggV2 is represented in its bi-allelic format.

Multi-allelic: where a single variant contains three or more observed alleles, counting the reference as one, therefore allowing for two or more variant alleles (heterozygous genotype example: 1/2)
Bi-allelic: where a variant contains two observed alleles, counting the reference as one, and therefore allowing for one variant allele (heterozygous genotypes are always: 0/1)

In variants that are decomposed, the INFO field is populated with the OLD_MULTIALLELIC tag which captures the original multi-allelic representation of the allele.

Decomposition example

Input:

chr1 12345678 . T A,G . .

Decomposed multiallelic variants:

chr1 12345678 . T A . . OLD_MULTIALLELIC=chr1:12345678:T/A/G
chr1 12345678 . T G . . OLD_MULTIALLELIC=chr1:12345678:T/A/G

Parsimony¶

A variant is parsimonious if it is represented in as few nucleotides as possible without an allele of length 0.

This step reduces the length of any variants to be parsimonious, while keeping the actual nucleotide change the same.

Parsimony example

Input:

chr1 12345678 . TAAA TAA . .

Parsimonious variant:

chr1 12345678 . TA T . .

Left alignment¶

A variant is left aligned if it is no longer possible to shift its position to the left while keeping the length of all its alleles constant.

This step shifts all variants to the left.

Left aligned example

Input:

chr1 12345678 . AAAA A . .

Left aligned variant:

chr1 12345670 . TAAA T . .

Decomposition of bi-allelic block substitutions (MNPs)¶

This step decomposes bi-allelic block substitutions (MNPs - Multi-Nucleotide Polymorphisms) into its constituent SNPs.

SNP: The reference and alternate sequences are of length 1 and the base nucleotide is different from one another.
MNP: The reference and alternate sequences are of the same length and have to be greater than 1 and all nucleotides in the sequences differ from one another

SNPs derived from the decomposition of MNPs are flagged with the OLD_CLUMPED tag in the INFO field which captures the original MNP representation of the variant.

Decomposition example

Input:

chr1 12345678 . TA CG . .

Decomposed biallelic blocks:

chr1 12345678 . T C . . OLD_CLUMPED=chr1:12345678:TA/CG
chr1 12345679 . A G . . OLD_CLUMPED=chr1:12345678:TA/CG

Code¶

The code below shows the normalisation procedure applied to each VCF output chunk from the aggregation by gvcf genotyper.

vt decompose -s ${input} | # Decomposition
vt normalize -n -w 10000 -r ${reference} - | # Parsimony and left alignment
vt decompose_blocksub - # Decomposition of bi-allelic block substitutions

Variant representation¶

There is no exact solution to variant normalisation and decomposition. Many downstream tools rely on variants being represented in their bi-allelic format. Bi-allelic representation also allows for easier allelic comparisons between call sets. You should be aware, however, of the implications the decomposition has in handling multi-allelic variants, as partial genotypes (e.g. "./0", "./1") are generated. From vt: Information is generally lost after vertically decomposing a variant, so care should be taken in interpreting the resultant values.

Partial genotypes¶

The normalisation procedure applied by vt decomposes all multi-allelic variants into their bi-allelic representations.

Definitions:

Multi-allelic: where a single variant contains three or more observed alleles, counting the reference as one, therefore allowing for two or more variant alleles (heterozygous genotype example: 1/2)
Bi-allelic: where a variant contains two observed alleles, counting the reference as one, and therefore allowing for one variant allele (heterozygous genotypes are always: 0/1)

Many downstream tools rely on variants being represented in their bi-allelic format. Bi-allelic representation also allows for easier allelic comparisons between call sets.

From vt: Information is generally lost after vertically decomposing a variant, so care should be taken in interpreting the resultant values.

The OLD_MULTIALLELIC INFO tag¶

Multi-allelic variants that have been decomposed into their bi-allelic representations are identified by the OLD_MULTIALLELIC tag in the INFO field of aggV2.

Worked example¶

Below is a worked example of how multi-allelic variants are represented in their bi-allelic format:

Pre-decomposed (multi-allelic representation)¶

CHROM	POS	ID	REF	ALT	QUAL	FILTER	INFO	FORMAT	SAMPLE 1	SAMPLE 2
chr1	3759889	.	TA	TAA,TAAA,T	.	PASS	.	GT	`1/2`	`0/0`

There are four alleles of this variant (including the REF allele). Sample 1 has genotype 1/2 (TAA, TAAA). Sample 2 has genotype 0/0 (TA, TA).

Post-decomposition (bi-allelic representation)¶

CHROM	POS	ID	REF	ALT	QUAL	FILTER	INFO	FORMAT	SAMPLE 1	SAMPLE 2
chr1	3759889	.	TA	TAA	.	PASS	OLD_MULTIALLELIC=1:3759889:TA/TAA/TAAA/T	GT	`1/.`	`0/0`
chr1	3759889	.	TA	TAAA	.	PASS	OLD_MULTIALLELIC=1:3759889:TA/TAA/TAAA/T	GT	`./1`	`0/0`
chr1	3759889	.	TA	T	.	PASS	OLD_MULTIALLELIC=1:3759889:TA/TAA/TAAA/T	GT	`./.`	`0/0`

The three ALT alleles have been decomposed into three separate lines; where each line represents one of the ALT alleles against the same REF allele.
The INFO field is populated with the OLD_MULTIALLELIC tag which captures the original multi-allelic representation of the allele.
Partial genotypes are generated for Sample 1 for this variant - who has genotype TAA, TAAA. This is because:
- For the first bi-allelic variant (TA, TAA), Sample 1 has the TAA ALT allele but not the TA REF allele (no information present for this allele) - so is therefore represented by the partial genotype: 1/.
- For the second bi-allelic variant (TA, TAAA), Sample 1 has the TAAA ALT allele but not the TA REF allele (no information present for this allele) - so is therefore represented by the partial genotype: ./1
- For the third bi-allelic variant (TA, T), Sample 1 has neither the T ALT allele nor the TA REF allele (no information present for these alleles) - so is therefore represented by the partial genotype: ./.
Partial genotypes are not represented for Sample 2 for this variant - who has genotype TA, TA. This is because:
- For all bi-allelic variants, Sample 2 is homozygous for the TA REF allele - so is always represented by the full genotype: 0/0

FORMAT field inheritance¶

The per-sample FORMAT tags (for example: GT- genotype, GQ - genotype quality, DP - depth, AD - allelic depth, PL - genotype likelihoods) are also vertically decomposed and follow two rules:

The GQ and DP tags are always identical for a given genotype when the variant is decomposed
The AD and PL tags are always representative of the two specific alleles per bi-allelic variant

This is shown in the examples below showing a single sample:

Example 1: he sample is homozygous for the C REF allele for the chr18:7311133:C/A/T variant¶

The sample genotype will always be 0/0 for all bi-allelic representations of this variant
The GQ and DP tags are identical for all bi-allelic representations of this variant
The AD and PL tags are identical for all bi-allelic representations of this variant as no information is lost after decomposition

Chrom	Pos	Ref	Alt	OLD_MULTIALLELIC	GT	FT	GQ	GQX	DP	DPF	AD	PL
chr18	7311133	C	A	chr18:7311133:C/A/T	0/0	.	56	.	27	0	27,0	0,255,255
chr18	7311133	C	T	chr18:7311133:C/A/T	0/0	.	56	.	27	0	27,0	0,255,255

Example 2: the sample is heterozygous (T/C) for the chr18:7365195:T/C/G variant¶

The sample genotype is 0/1 for the T/C bi-allelic variant but partial (0/.) for the T/G bi-allelic variant as no information is present for the G allele
The GQ and DP tags are identical for all bi-allelic representations of this variant
The AD and PL tags are representative of the two specific alleles per bi-allelic variant (no depth in AD for the G allele, and PL set to 255 for the T/G and G/G genotypes)

Chrom	Pos	Ref	Alt	OLD_MULTIALLELIC	GT	FT	GQ	GQX	DP	DPF	AD	PL
chr18	7365195	T	C	chr18:7365195:T/C/G	0/1	PASS	200	46	31	3	14,17	232,0,197
chr18	7365195	T	G	chr18:7365195:T/C/G	0/.	PASS	200	46	31	3	14,0	232,255,255

Example 3: the sample is homozygous (A/A) for the chr18:7403330:G/A/C variant¶

The sample genotype is 1/1 for the G/A bi-allelic variant but partial (./.) for the G/C bi-allelic variant as no information is present for the G or C allele
The GQ and DP tags are identical for all bi-allelic representations of this variant
The AD and PL tags are representative of the two specific alleles per bi-allelic variant (no depth in AD for the G pr C allele, and PL set to 255 for the G/C and C/C genotypes)

Chrom	Pos	Ref	Alt	OLD_MULTIALLELIC	GT	FT	GQ	GQX	DP	DPF	AD	PL
chr18	7403330	G	A	chr18:7403330:G/A/C	1/1	PASS	30	17	11	0	0,11	218,33,0
chr18	7403330	G	C	chr18:7403330:G/A/C	./.	PASS	30	17	11	0	0,0	218,255,255

Summary¶

Bi-allelic variants derived from the same multi-allelic variant are identified by the OLD_MULTIALLELIC tag in the INFO field. The tag is present in all bi-allelic representations of the respective multi-allelic variant.
Vertical decomposition results in partial genotypes:
- 0/0 sample genotypes will always be decomposed to 0/0 for remaining bi-allelic variants of the same OLD_MULTIALLELIC tag
- 0/1 sample genotypes will always be decomposed to 0/. for remaining bi-allelic variants of the same OLD_MULTIALLELIC tag
- 1/1 sample genotypes will always be decomposed to ./. for remaining bi-allelic variants of the same OLD_MULTIALLELIC tag
The GQ and DP tags are always identical for a given genotype when the variant is decomposed; whereas the AD and PL tags are always representative of the two specific alleles per bi-allelic variant

If deemed absolutely necessary, you may want to post-process the partial genotypes like 1/. to the best guess genotype based on the PL values and recompute fields that involves alleles after decomposition.

Variant duplication and multi-nucleotide polymorphisms (MNPs)¶

A duplicated variant is a variant line with the same CHROM, POS, REF, and ALT that is represented more than once in aggV2. It was estimated that approximately 0.02% of the variants in the dataset are formed of duplicated variants.

There is no exact solution to this issue and it is important to handle duplicated variants with care as their allele frequencies might be affected.

Duplications arise from the decomposition of MNPs (Multi-Nucleotide Polymorphisms) into their constitutive SNP (Single-Nucleotide Polymorphisms) representations by vt (vt decompose_blocksub). This step is carried out post-aggregation.

SNPs derived from the decomposition of MNPs do not combine/merge with canonical SNPs (not derived from MNPs). This is what causes the duplication of lines. A single variant many be duplicated many times if the MNP is long and there are many canonical SNP variants.

Definitions

SNP: The reference and alternate sequences are of length 1 and the base nucleotide is different from one another.

MNP: The reference and alternate sequences are of the same length and have to be greater than 1 and all nucleotides in the sequences differ from one another

The OLD_CLUMPED INFO tag¶

SNPs derived from the decomposition of MNPs are flagged with the OLD_CLUMPED tag in the INFO field.

Worked example¶

Pre-decomposed MNP for a single sample:

CHROM	POS	ID	REF	ALT	QUAL	FILTER	INFO	FORMAT	SAMPLE 1
20	763837	.	CA	TG	.	PASS	AC=1;AN=2	GT	0/1

Decomposed MNP for a single-sample:

The CA > TG MNP has been decomposed into its constitutive SNPs: C > T and A > G.

The INFO filed has been populated with the OLD_CLUMPED tag - which keeps track of the original MNP that was decomposed.

CHROM	POS	ID	REF	ALT	QUAL	FILTER	INFO	FORMAT	SAMPLE 1
20	763837	.	C	T	.	PASS	AC=1;AN=2;OLD_CLUMPED=20:763837:CA/TG	GT	0/1
20	763838	.	A	G	.	PASS	AC=1;AN=2;OLD_CLUMPED=20:763837:CA/TG	GT	0/1

Pre-decomposed MNP in multi-sample:

Sample 1 is heterozygous for the CA > TG MNP.
Sample 2 is heterozygous for the A > G SNP.

Chromosomes	POS	ID	REF	ALT	QUAL	FILTER	INFO	FORMAT	SAMPLE 1	SAMPLE 2
20	763837	.	CA	TG	.	PASS	AC=1;AN=4	GT	0/1	0/0
20	763837	.	A	G	.	PASS	AC=1;AN=4	GT	0/0	0/1

**Decomposed MNP in muli-sample: **

The CA > TG MNP in Sample 1 has been decomposed into its constitutive SNPs: C > T and A > G. The INFO filed has been populated with the OLD_CLUMPED tag - which keeps track of the original MNP that was decomposed.

No change occurs to the A > G SNP for Sample 2.

SNPs derived from the decomposition of MNPs do not combine/merge with canonical SNPs (not derived from MNPs).

Therefore a duplicated variant (identical CHROM, POS, REF, ALT) is created for the A > G SNP.

These can be differentiated using the OLD_CLUMPED INFO tag, as this is populated when an MNP is decomposed, but is empty (.) for canonical SNPs.

CHROM	POS	ID	REF	ALT	QUAL	FILTER	INFO	FORMAT	SAMPLE 1	SAMPLE 2
20	763837	.	C	T	.	PASS	AC=1;AN=4;OLD_CLUMPED=20:763837:CA/TG	GT	0/1	0/0
20	763838	.	A	G	.	PASS	AC=1;AN=4;OLD_CLUMPED=20:763837:CA/TG	GT	0/1	0/0
20	763838	.	A	G	.	PASS	AC=1;AN=2	GT	0/1	0/1

Identifying unique variants¶

As mentioned, only ~0.02% of the variants in the dataset are duplicated (identical CHROM, POS, REF, ALT).

All variants are completely unique however if the following fields are concatenated per variant:

CHROM, POS, REF, ALT, INFO/OLD_MULTIALLELIC, INFO/OLD_CLUMPED

Variants with allele count of 0¶

There are a few instances of variants in aggV2 that have an allele count (AC) of zero. There are two reasons as to why this is observed:

Participants that withdraw from the programme are removed from the dataset post-aggregation. Though their genotypes are removed, their variants are kept, so to avoid confusion of variant lines that may contain partial genotypes. Such variants will have an AC of 0, as the AC is calculated post-removal of withdrawn participants.
In the single sample gVCFs, certain variants are 'forced-genotyped' meaning that a variant call is made even if no variant exists - i.e. the sample genotype will not be in a REF BLOCK but be coded as 0/0. Forced-genotype variants are preserved in aggV2. Therefore if all samples are 0/0 for a particular forced-genotyped variant, then the AC of that variant will be 0.

Maximum alternate alleles per variant¶

In the gVCF aggregation process by gvcf genotyper, multi-allelic variants with more than 50 alternate alleles are discarded and not included in aggV2.

Variants within deletions¶

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:

CHROM	POS	REF	ALT	GT	Description
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) 2bp 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).

CHROM	POS	REF	ALT	GT	Description
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).

In aggV2, the haploid call for that sample-genotype is carried over as haploid from the single sample gVCF.

Help and support¶

Please reach out via the Genomics England Service Desk for any issues related to the aggV2 aggregation or companion datasets, including "aggV2" in the title/description of your inquiry.