Supported TR Genotypers

TRTools currently supports 6 tandem repeat genotypers. It also supports the Beagle imputation software (see below). We summarize them in the first table and provide some basic parameters of their functionality in the second. For more information on a genotyper, please see its website linked below.

Genotyper (version tested)	Use case notes
AdVNTR (v1.3.3)	Infers allele lengths. May alternatively identify putative frameshift mutations within VNTRs (6+bp repeat units). Designed for targeted genotyping of VNTRs. May be run on large panels of TRs but is compute-intenstive.
ExpansionHunter (v3.2.2)	Handles repeats with structures such as interruptions or nearby repeats. Designed for targeted genotyping of expansions at known pathogenic TRs but may be run genome-wide on short and expanded TRs using a custom TR panel.
GangSTR (2.4.4)	Designed for genome-wide genotyping of short or expanded TRs.
HipSTR (v0.6.2)	Designed for genome-wide genotyping of STR (1-6bp repeat units) alleles shorter than the read length. Can phase repeats with SNPs.
PopSTR (v2.0)	Designed for genome-wide genotyping of short or expanded TRs.
LongTR (v1.0)	Designed for genome-wide genotyping of STRs and VNTRs from long reads.

Genotyper (version tested)	Repeat unit lengths	Alleles longer than reads?	Allele type inferred	# TRs in reference	Sequencing technology	# Samples at a time
AdVNTR (v1.3.3)	6-100bp	No	Length, frameshifts	158,522 (genic hg19)	Illumina, PacBio	Single
ExpansionHunter (v3.2.2)	1-6bp. Can handle complex repeat structures specified by regular expressions	Yes	Length	25 (hg19)	PCR-free Illumina	Single
GangSTR (2.4.4)	1-20bp	Yes	Length	829,233 (hg19)	Paired-end Illumina	Many
HipSTR (v0.6.2)	1-9bp	No	Length, sequence	1,620,030 (hg19)	Illumina	Many
PopSTR (v2.0)	1-6bp	Yes	Length	540,1401 (hg38)	Illumina	Many
LongTR (v1.0)	1+bp	No	Length, sequence	No ref provided	PacBio HiFi, ONT	Many

Since each of these tools take as input a list of TRs to genotype, they could also be used on custom panels of TR loci. Tool information and reference panel numbers shown above are based on downloads from the github repository of each tool as of July 2, 2020.

TRTools can be extended to support other genotypers that generate VCF files. We welcome community contributions to help support them. If that interests you, please see Contributing for more information.

Beagle

The Beagle software can take genotypes called by a TR genotyper in a set of reference samples and impute them into other samples that do not have directly genotyped TRs. TRTools supports TR genotypes produced by any of the above genotypers and then imputed into other samples with Beagle except for PopSTR genotypes. For each tool in this tool suite, unless it’s docs specifically say otherwise, that tool can be used on Beagle VCFs as if those VCFs were produced directly by the underlying TR genotyper, with no additional flags or arguments needed, as long as the steps below were followed to make sure the Beagle VCF is properly formatted.

Notes and caveats:

• Beagle provides phased best-guess genotypes for each imputed sample at each TR locus. When run with the ap or gp flags Beagle will also output probabilities for each possible allele/genotype, respectively. The allele probabilities can be used to compute genotype dosages that take into account imputation uncertainty. The annotaTR utility described below can be used to compute dosages based on the AP fields. These can also be accessed from the TR Harmonizer TRRecord.GetDosages function.

• At each locus Beagle returns the most probable phased genotype. This will often but not always correspond to the most probable unphased genotype. For instance, it is possible that P(A|A) > P(A|B) and P(A|A) > P(B|A), but P(A/A) = P(A|A) < P(A|B) + P(B|A) = P(A/B). Similarly, it is possible that P(A|B) > P(C|D) and P(A|B) > P(D|C), but P(A/B) = P(A|B) + P(B|A) < P(C|D) + P(D|C) = P(C/D). TRTools currently does not take this into account and just uses the phased genotypes returned by Beagle. If you would like to add functionality to support this, feel free to submit PRs to help TRTools take this into account (see the Contributing docs).

• For callers which return sequences, not just lengths (e.g. HipSTR), if there are loci with multiple plausible sequences of the same length, then it’s possible that the most probable genotype returned by Beagle does not have the most probable length. For example, the following could be true of a single haplotype: Len(S_1) = L_1, Len(S_2) = L_1, Len(S_3) = L_2 and P(S_1) < P(S_3), P(S_2) < P(S_3) but P(S_3) < P(S_1) + P(S_2).

See here for example commands for running Beagle to impute TRs. Some additional notes:

• VCF files for the samples being imputed into must include genotyped loci (typically SNPs) that are also genotyped in the reference samples. This allows those samples to be ‘matched’ with samples in the reference.

• The reference samples must be phased and also not contain any missing genotypes. Possible methods for dealing with that include removing loci with missing genotypes or using imputation to impute the missing genotypes prior to imputing the TRs. The reference panel at the link above has already been phased/imputed and should work directly as input to Beagle.

The VCFs that Beagle outputs need to be preprocessed before use by TRTools to contain required INFO fields. The annotaTR utility provided by TRTools can be used to add this information as well as compute dosages, with the option to output either VCF or PGEN format. (As of Aug 2024, we had only provided the script trtools_prep_beagle_vcf.sh which is still available and can also be used to add the INFO annotations but cannot write to PGEN files or compute dosages). After running either of those tools to annotate Beagle VCFs, the files should be usable by any of the tools in TRTools.

In case of error, it may be useful to know what steps those tools attempt to perform:

• Copy over source and command meta header lines from the reference panel to the imputed VCF so that it is clear which genotyper’s syntax is being used to represent the STRs in the VCF.

• Copy over contig and ALT lines which is required for downstream tools including mergeSTR and is good practice to include in the VCF header.

• Annotate each STR with the necessary INFO fields from the reference panel that Beagle dropped from the imputed VCF.

• Removes the non-TR loci (identified as those loci not having TR-specific INFO fields) so that the final output file contains only TRs.