example.md

Introduction

A brief overview of the steps needed to call de novo tandem repeat mutations using TRGT-denovo given TRGT output.

Prerequisites

• TRGT binary
• TRGT-denovo binary
• Repeat definition files are available in this Zenodo repository and definitions of known pathogenic repeats are also available here (always the same sites or a subset of those used with TRGT)
• Aligned HiFi data of a family trio (father, mother, and child)
• The reference genome used for read alignment

Calling de novo tandem repeat mutations

Given the following data:

• Reference genome reference.fasta
• Repeat definition file repeat.bed.
• Aligned sequencing data of the family (father, mother, and son respectively) sample_F.bam, sample_M.bam, and sample_S.bam.

Data pre-processing

All data must first be genotyped by TRGT:

./trgt --genome reference.fasta \
       --repeats repeat.bed \
       --reads sample_F.bam \
       --output-prefix sample_F \
       --karyotype XY

./trgt --genome reference.fasta \
       --repeats repeat.bed \
       --reads sample_M.bam \
       --output-prefix sample_M \
       --karyotype XX

./trgt --genome reference.fasta \
       --repeats repeat.bed \
       --reads sample_S.bam \
       --output-prefix sample_S \ 
       --karyotype XY

TRGT outputs the genotyped repeat sites in a VCF file stored in prefix.vcf.gz and the spanning reads that were used to genotype each site (that fully span the repeat sequences) stored in prefix.spanning.bam. TRGT-denovo requires sorted BAM and VCF data, hence you will need to sort and index the output VCF and BAM files. For each family member this involves:

VCF sorting

bcftools sort -Ob -o sample_F.sorted.vcf.gz sample_F.vcf.gz
bcftools index sample_F.sorted.vcf.gz

BAM sorting

samtools sort -o sample_F.spanning.sorted.bam sample_F.spanning.bam
samtools index sample_F.spanning.sorted.bam

Such that you end up with sample_F.sorted.vcf.gz, sample_F.spanning.sorted.bam, sample_M.sorted.vcf.gz, sample_M.spanning.sorted.bam, sample_S.sorted.vcf.gz, sample_S.spanning.sorted.bam (and their associated .bam.bai and .vcf.gz.csi indices).

Running TRGT-denovo

With all preprocessing completed, we can call de novo repeat expansion mutations using TRGT-denovo from the sample data. Note that family members are supplied by their common prefix of spanning.sorted.bam and sorted.vcf.gz, i.e., sample_F, sample_M, and sample_S and path if not running TRGT-denovo in the same directory as the data:

./TRGT-denovo trio --reference reference.fasta \
              --bed repeat.bed \
              --father sample_F \
              --mother sample_M \
              --child sample_S \
              --out out.tsv

For further interpretation of TRGT-denovo output see here, additionally scripts/python/trio_analysis.ipynb shows an example of doing basic analysis given TRGT-denovo trio output to select candidate de novo calls.