Data processing and visualisation for WGS-PGT (whole genome sequencing - preimplantation genetic testing).
Note: the included bash scripts are optimised for computation on a high performance computing cluster running a SLURM job scheduler. The specified resource requirements were optimised for the sequencing data generated for this project.
Sequencing output is processed via the following steps (scripts not included here)
- Demultiplexing
- FASTQC
- Alignment using BWA-MEM2
- BAMQC
- GATK Joint-genotyping using GATK4
Family-wise batches (i.e. multisample vcf files) are generated using gatk-haplotype-joint calling. The resulting vcf file was analysed by Haplarithmisis.
GATK4 from Broad Institute is available here
These are all relevant R scripts to process whole genome sequencing data using Haplarithmisis for WGS-PGT
-
MetaInfo
input: CSV file with family information (example attached)- Sample ID (Sample ID of each of the family members / embryos)
- Family number (PGT + familynumber)
- PGT (diagnostics / research)
- Sample Status (E = Embryo, Combination of U = unaffected or A = affected and family member: F = Father, M = Mother, S = Sibling, GF = GrandFather, GM = GrandMother)
- Family interval (chr_startposition_endposition_parent, example: chr2_1001_1002_Pat)
- Family second interval (in case of a compound heterozygous mutation, if not applicable: Non Defined (ND))
- Family indication (GENE + "_ PGT")
- Family Dnr (prefix D"year of analysis/number")
input: PGT config file (.txt) with path to scripts, samplesheet and default parameters
- default parameters: Win=10, gammaBAF=10, bin=10000, Window=22, gammaSC=300, gammaMC=50, ExtInt=1, plateau=100, gtypemodulator_window=10000
- Subsampling to desired target coverage
- Visualisation of lab step timings
- coverage metrics from qualimap
- Extract qualimap output
- Visualisation coverage metrics
- Visualisation of Mendelian inconsistency for validation and pilot per subsampled target coverage and validation per chromosome
- Visualisation of Haplotype concordance for pilot at subsampled target coverages and validation
- liftover coordinates from onePGT output
- informative SNP binning and chromosome heatmap visualisation - see PGT-SR folder for chromosome coordinate scripts.
- input: CSV file with family information (example attached for parents-only haplarithmisis)
- Haplarithm plotting with chromosome ideogram - see PGT-SR folder for ideogram coordinate scripts.
Embryo trophectoderm biopsy (and parental/reference) data was processed with the following steps:
- The data were processed as per the PGT-M processing up to and including haplarithmisis.
- Deep (30-40X) sequenced data was subsampled as per PGT-M and the (segmented) logRs were plotted
- breakpoint analysis using Manta
- Relevant breakpoint extraction
- Custom visualisation of haplarithms including breakpoint information & chromosome schematics + generation of chromosome fill / outline coordinates for normal and affected
- Visualisation of copy number variation from VeriSeq output.
Embryo trophectoderm biopsy WGS data was processed with the following steps:
- The data were processed as per the PGT-M processing up to and including the alignment step. (alignment was done to the Hg38 reference genome including the mitochondrial "chromosome")
- (Samples that were deep sequenced (30-40X) were subsampled with the aforementioned procedure.
- Mitochondrial DNA coverage calculation & visualisation
- Heteroplasmy level calculation
MITOMAP: https://www.mitomap.org/foswiki/bin/view/MITOMAP/ConfirmedMutations