This vignette shows how to process long-read PacBio HiFi variant calls from a validated trio (HG002–HG003–HG004) and prepare them for UPDhmm analysis.

Data source

Ashkenazi trio (GIAB, NIST) – PacBio HiFi Revio, DeepVariant calls (GRCh38).

1. Download phased VCFs for each individual


# Proband (HG002)
wget ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/AshkenazimTrio/analysis/PacBio_HiFi-Revio_20231031/pacbio-wgs-wdl_germline_20231031/HG002.GRCh38.deepvariant.phased.vcf.gz
wget ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/AshkenazimTrio/analysis/PacBio_HiFi-Revio_20231031/pacbio-wgs-wdl_germline_20231031/HG002.GRCh38.deepvariant.phased.vcf.gz.tbi

# Father (HG003)
wget ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/AshkenazimTrio/analysis/PacBio_HiFi-Revio_20231031/pacbio-wgs-wdl_germline_20231031/HG003.GRCh38.deepvariant.phased.vcf.gz
wget ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/AshkenazimTrio/analysis/PacBio_HiFi-Revio_20231031/pacbio-wgs-wdl_germline_20231031/HG003.GRCh38.deepvariant.phased.vcf.gz.tbi

# Mother (HG004)
wget ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/AshkenazimTrio/analysis/PacBio_HiFi-Revio_20231031/pacbio-wgs-wdl_germline_20231031/HG004.GRCh38.deepvariant.phased.vcf.gz
wget ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/AshkenazimTrio/analysis/PacBio_HiFi-Revio_20231031/pacbio-wgs-wdl_germline_20231031/HG004.GRCh38.deepvariant.phased.vcf.gz.tbi

2. Merge individual VCFs into a trio VCF

bcftools merge \
  -O z \
  -o trio_HiFi_GRCh38_phased.vcf.gz \
  HG002.GRCh38.deepvariant.phased.vcf.gz \
  HG003.GRCh38.deepvariant.phased.vcf.gz \
  HG004.GRCh38.deepvariant.phased.vcf.gz

bcftools index trio_HiFi_GRCh38_phased.vcf.gz

3. Filter variants for UPDhmm input

The following filtering steps are applied:

keep only biallelic variants
remove sites where all trio members are reference (0/0 or 0|0)
remove sites where all trio members are missing (./. or .|.)

bcftools view \
  -m2 -M2 \
  -e 'COUNT(GT="0/0" || GT="0|0")==3 || COUNT(GT="./." || GT=".|.")==3' \
  -O z \
  -o trio_HiFi_GRCh38_phased_biallelic_nonref_nomissing.vcf.gz \
  trio_HiFi_GRCh38_phased.vcf.gz

bcftools index trio_HiFi_GRCh38_phased_biallelic_nonref_nomissing.vcf.gz

4. UPDhmm analysis in R

library(UPDhmm)
library(VariantAnnotation)

vcf <- readVcf(
  "trio_HiFi_GRCh38_phased_biallelic_nonref_nomissing.vcf.gz"
)

vcf_check <- vcfCheck(
  <!-- vcf, -->
  proband = "HG002",
  father  = "HG003",
  mother  = "HG004"
)

events <- calculateEvents(
  vcf_check,
  add_ratios = TRUE
)

Session Info

sessionInfo()

## R version 4.5.2 (2025-10-31)
## Platform: x86_64-pc-linux-gnu
## Running under: Ubuntu 24.04.3 LTS
## 
## Matrix products: default
## BLAS:   /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3 
## LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.26.so;  LAPACK version 3.12.0
## 
## locale:
##  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
##  [3] LC_TIME=en_US.UTF-8        LC_COLLATE=C              
##  [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
##  [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
##  [9] LC_ADDRESS=C               LC_TELEPHONE=C            
## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       
## 
## time zone: Etc/UTC
## tzcode source: system (glibc)
## 
## attached base packages:
## [1] stats4    stats     graphics  grDevices utils     datasets  methods  
## [8] base     
## 
## other attached packages:
##  [1] karyoploteR_1.37.0          regioneR_1.43.0            
##  [3] VariantAnnotation_1.57.1    Rsamtools_2.27.0           
##  [5] Biostrings_2.79.4           XVector_0.51.0             
##  [7] SummarizedExperiment_1.41.1 Biobase_2.71.0             
##  [9] GenomicRanges_1.63.1        IRanges_2.45.0             
## [11] S4Vectors_0.49.0            Seqinfo_1.1.0              
## [13] MatrixGenerics_1.23.0       matrixStats_1.5.0          
## [15] BiocGenerics_0.57.0         generics_0.1.4             
## [17] dplyr_1.2.0                 UPDhmm_1.7.1               
## [19] BiocStyle_2.39.0           
## 
## loaded via a namespace (and not attached):
##  [1] DBI_1.2.3                bitops_1.0-9             gridExtra_2.3           
##  [4] rlang_1.1.7              magrittr_2.0.4           biovizBase_1.59.0       
##  [7] compiler_4.5.2           RSQLite_2.4.6            GenomicFeatures_1.63.1  
## [10] png_0.1-8                vctrs_0.7.1              ProtGenerics_1.43.0     
## [13] stringr_1.6.0            pkgconfig_2.0.3          crayon_1.5.3            
## [16] fastmap_1.2.0            backports_1.5.0          rmarkdown_2.30          
## [19] UCSC.utils_1.7.1         bit_4.6.0                xfun_0.56               
## [22] cachem_1.1.0             cigarillo_1.1.0          GenomeInfoDb_1.47.2     
## [25] jsonlite_2.0.0           blob_1.3.0               DelayedArray_0.37.0     
## [28] BiocParallel_1.45.0      parallel_4.5.2           cluster_2.1.8.2         
## [31] R6_2.6.1                 bslib_0.10.0             stringi_1.8.7           
## [34] RColorBrewer_1.1-3       bezier_1.1.2             rtracklayer_1.71.3      
## [37] rpart_4.1.24             jquerylib_0.1.4          Rcpp_1.1.1              
## [40] knitr_1.51               base64enc_0.1-6          Matrix_1.7-4            
## [43] nnet_7.3-20              tidyselect_1.2.1         rstudioapi_0.18.0       
## [46] dichromat_2.0-0.1        abind_1.4-8              yaml_2.3.12             
## [49] codetools_0.2-20         curl_7.0.0               lattice_0.22-9          
## [52] tibble_3.3.1             KEGGREST_1.51.1          S7_0.2.1                
## [55] evaluate_1.0.5           foreign_0.8-91           pillar_1.11.1           
## [58] BiocManager_1.30.27      checkmate_2.3.4          RCurl_1.98-1.17         
## [61] ensembldb_2.35.0         ggplot2_4.0.2            scales_1.4.0            
## [64] glue_1.8.0               lazyeval_0.2.2           Hmisc_5.2-5             
## [67] maketools_1.3.2          tools_4.5.2              BiocIO_1.21.0           
## [70] data.table_1.18.2.1      sys_3.4.3                BSgenome_1.79.1         
## [73] GenomicAlignments_1.47.0 buildtools_1.0.0         XML_3.99-0.22           
## [76] grid_4.5.2               AnnotationDbi_1.73.0     colorspace_2.1-2        
## [79] htmlTable_2.4.3          restfulr_0.0.16          Formula_1.2-5           
## [82] cli_3.6.5                HMM_1.0.2                S4Arrays_1.11.1         
## [85] AnnotationFilter_1.35.0  gtable_0.3.6             sass_0.4.10             
## [88] digest_0.6.39            SparseArray_1.11.10      rjson_0.2.23            
## [91] htmlwidgets_1.6.4        farver_2.1.2             memoise_2.0.1           
## [94] htmltools_0.5.9          lifecycle_1.0.5          httr_1.4.8              
## [97] bit64_4.6.0-1            bamsignals_1.43.0