# Long reads VCF Preprocessing User Guide

Marta Sevilla Porras1,2* and Carlos Ruiz Arenas3**

1Universitat Pompeu Fabra (UPF)
2Centro de Investigación Biomédica en Red (CIBERER)
3Universidad de Navarra (UNAV)

*marta.sevilla@upf.edu
**cruizarenas@unav.es

#### 1 June 2026

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 4.6.0 Patched (2026-05-01 r89994)
    ## Platform: aarch64-apple-darwin23
    ## Running under: macOS Tahoe 26.3.1
    ##
    ## Matrix products: default
    ## BLAS:   /Library/Frameworks/R.framework/Versions/4.6/Resources/lib/libRblas.0.dylib
    ## LAPACK: /Library/Frameworks/R.framework/Versions/4.6/Resources/lib/libRlapack.dylib;  LAPACK version 3.12.1
    ##
    ## locale:
    ## [1] C/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
    ##
    ## time zone: America/New_York
    ## tzcode source: internal
    ##
    ## attached base packages:
    ## [1] stats4    stats     graphics  grDevices utils     datasets  methods
    ## [8] base
    ##
    ## other attached packages:
    ##  [1] karyoploteR_1.39.0          regioneR_1.45.0
    ##  [3] VariantAnnotation_1.59.0    Rsamtools_2.29.0
    ##  [5] Biostrings_2.81.2           XVector_0.53.0
    ##  [7] SummarizedExperiment_1.43.0 Biobase_2.73.1
    ##  [9] GenomicRanges_1.65.0        IRanges_2.47.2
    ## [11] S4Vectors_0.51.3            Seqinfo_1.3.0
    ## [13] MatrixGenerics_1.25.0       matrixStats_1.5.0
    ## [15] BiocGenerics_0.59.6         generics_0.1.4
    ## [17] dplyr_1.2.1                 UPDhmm_1.9.0
    ## [19] BiocStyle_2.41.0
    ##
    ## loaded via a namespace (and not attached):
    ##   [1] DBI_1.3.0                bitops_1.0-9             gridExtra_2.3
    ##   [4] rlang_1.2.0              magrittr_2.0.5           biovizBase_1.61.0
    ##   [7] otel_0.2.0               compiler_4.6.0           RSQLite_3.53.1
    ##  [10] GenomicFeatures_1.65.0   png_0.1-9                vctrs_0.7.3
    ##  [13] ProtGenerics_1.45.0      stringr_1.6.0            pkgconfig_2.0.3
    ##  [16] crayon_1.5.3             fastmap_1.2.0            magick_2.9.1
    ##  [19] backports_1.5.1          rmarkdown_2.31           UCSC.utils_1.9.0
    ##  [22] tinytex_0.59             bit_4.6.0                xfun_0.58
    ##  [25] cachem_1.1.0             cigarillo_1.3.0          GenomeInfoDb_1.49.1
    ##  [28] jsonlite_2.0.0           blob_1.3.0               DelayedArray_0.39.3
    ##  [31] BiocParallel_1.47.0      parallel_4.6.0           cluster_2.1.8.2
    ##  [34] R6_2.6.1                 stringi_1.8.7            bslib_0.11.0
    ##  [37] RColorBrewer_1.1-3       bezier_1.1.2             rtracklayer_1.73.0
    ##  [40] rpart_4.1.27             jquerylib_0.1.4          Rcpp_1.1.1-1.1
    ##  [43] bookdown_0.46            knitr_1.51               base64enc_0.1-6
    ##  [46] BiocBaseUtils_1.15.1     Matrix_1.7-5             nnet_7.3-20
    ##  [49] tidyselect_1.2.1         rstudioapi_0.18.0        dichromat_2.0-0.1
    ##  [52] abind_1.4-8              yaml_2.3.12              codetools_0.2-20
    ##  [55] curl_7.1.0               lattice_0.22-9           tibble_3.3.1
    ##  [58] KEGGREST_1.53.0          S7_0.2.2                 evaluate_1.0.5
    ##  [61] foreign_0.8-91           pillar_1.11.1            BiocManager_1.30.27
    ##  [64] checkmate_2.3.4          RCurl_1.98-1.18          ensembldb_2.37.1
    ##  [67] ggplot2_4.0.3            scales_1.4.0             glue_1.8.1
    ##  [70] lazyeval_0.2.3           Hmisc_5.2-5              tools_4.6.0
    ##  [73] BiocIO_1.23.3            data.table_1.18.4        BSgenome_1.81.0
    ##  [76] GenomicAlignments_1.49.0 XML_3.99-0.23            grid_4.6.0
    ##  [79] colorspace_2.1-2         AnnotationDbi_1.75.0     htmlTable_2.5.0
    ##  [82] restfulr_0.0.16          Formula_1.2-5            cli_3.6.6
    ##  [85] HMM_1.0.2                S4Arrays_1.13.0          AnnotationFilter_1.37.0
    ##  [88] gtable_0.3.6             sass_0.4.10              digest_0.6.39
    ##  [91] SparseArray_1.13.2       rjson_0.2.23             htmlwidgets_1.6.4
    ##  [94] farver_2.1.2             memoise_2.0.1            htmltools_0.5.9
    ##  [97] lifecycle_1.0.5          httr_1.4.8               bit64_4.8.2
    ## [100] bamsignals_1.45.1