HiCool

Jacques Serizay

Contents

1 Processing sequencing Hi-C libraries with HiCool

The HiCool R/Bioconductor package provides an end-to-end interface to process and normalize Hi-C paired-end fastq reads into .(m)cool files.

1. The heavy lifting (fastq mapping, pairs parsing and pairs filtering) is performed by the underlying lightweight hicstuff python library (https://github.com/koszullab/hicstuff).
2. Pairs filering is done using the approach described in Cournac et al., 2012 and implemented in hicstuff.
3. cooler (https://github.com/open2c/cooler) library is used to parse pairs into a multi-resolution, balanced .mcool file. .(m)cool is a compact, indexed HDF5 file format specifically tailored for efficiently storing HiC-based data. The .(m)cool file format was developed by Abdennur and Mirny and published in 2019.
4. Internally, all these external dependencies are automatically installed and managed in R by a basilisk.utils conda environment.

The main processing function offered in this package is HiCool(). To process .fastq reads into .pairs & .mcool files, one needs to provide:

• The path to each fastq file (r1 and r2);
• The genome reference, as a path to a .fasta sequence file, a path to a pre-computed bowtie2 index or a supported ID character (hg38, mm10, dm6, R64-1-1, WBcel235, GRCz10, Galgal4);
• The restriction enzyme(s) used for Hi-C.

x <- HiCool(
    r1 = '<PATH-TO-R1.fq.gz>', 
    r2 = '<PATH-TO-R2.fq.gz>', 
    restriction = '<RE1(,RE2)>', 
    binning = "<minimum resolution>", 
    genome = '<GENOME_ID>'
)

Here is a concrete example of Hi-C data processing.

• Example fastq files are retrieved using the HiContactsData package.
• Two restriction enzymes are used (these are the enzymes used in the Arima Kit).
• The final .mcool file will have three levels of resolutions, from 1000bp to 8000bp.
• The data will be mapped on R64-1-1, the yeast genome reference.
• All output files will be placed in output/ directory.

library(HiCool)
hcf <- HiCool(
    r1 = HiContactsData::HiContactsData(sample = 'yeast_wt', format = 'fastq_R1'), 
    r2 = HiContactsData::HiContactsData(sample = 'yeast_wt', format = 'fastq_R2'), 
    restriction = 'DpnII,HinfI', 
    binning = 1000, 
    genome = 'R64-1-1', 
    output = './HiCool/'
)
#> HiCool :: Parsing HiCool arguments and checking files.
#> see ?HiContactsData and browseVignettes('HiContactsData') for documentation
#> loading from cache
#> see ?HiContactsData and browseVignettes('HiContactsData') for documentation
#> loading from cache
#> HiCool :: Recovering bowtie2 genome index from AWS iGenomes...
#> HiCool :: Processing .fastq files to .mcool format. This might take a while.
#> HiCool :: Initializing processing of fastq files [tmp folder: /tmp/RtmpjaJK5d/WRFG0S]...
#> HiCool :: Mapping fastq files...
#> HiCool :: Tidying up everything for you...
#> HiCool :: .fastq to .mcool processing done!
#> HiCool :: Check ./HiCool/folder to find the generated files
#> HiCool :: Generating HiCool report. This might take a while.
#> HiCool :: Report generated and available @ /tmp/Rtmp1xC6Rp/Rbuild2eb1fd6066ca29/HiCool/vignettes/HiCool/53e067b8d8def_7833^mapped-R64-1-1^WRFG0S.html
#> HiCool :: All processing successfully achieved. Congrats!

hcf
#> CoolFile object
#> .mcool file: ./HiCool//matrices/53e067b8d8def_7833^mapped-R64-1-1^WRFG0S.mcool 
#> resolution: 1000 
#> pairs file: ./HiCool//pairs/53e067b8d8def_7833^mapped-R64-1-1^WRFG0S.pairs 
#> metadata(3): log args stats
S4Vectors::metadata(hcf)
#> $log
#> [1] "./HiCool//logs/53e067b8d8def_7833^mapped-R64-1-1^WRFG0S.log"
#> 
#> $args
#> $args$r1
#> [1] "/home/biocbuild/.cache/R/ExperimentHub/53e067b8d8def_7833"
#> 
#> $args$r2
#> [1] "/home/biocbuild/.cache/R/ExperimentHub/53e069fb62ae_7834"
#> 
#> $args$genome
#> [1] "/tmp/RtmpjaJK5d/R64-1-1"
#> 
#> $args$binning
#> [1] "1000"
#> 
#> $args$restriction
#> [1] "DpnII,HinfI"
#> 
#> $args$iterative
#> [1] TRUE
#> 
#> $args$balancing_args
#> [1] " --min-nnz 10 --mad-max 5 "
#> 
#> $args$threads
#> [1] 1
#> 
#> $args$output
#> [1] "./HiCool/"
#> 
#> $args$exclude_chr
#> [1] "Mito|chrM|MT"
#> 
#> $args$keep_bam
#> [1] FALSE
#> 
#> $args$scratch
#> [1] "/tmp/RtmpjaJK5d"
#> 
#> $args$wd
#> [1] "/tmp/Rtmp1xC6Rp/Rbuild2eb1fd6066ca29/HiCool/vignettes"
#> 
#> 
#> $stats
#> $stats$nFragments
#> [1] 1e+05
#> 
#> $stats$nPairs
#> [1] 64761
#> 
#> $stats$nDangling
#> [1] 9266
#> 
#> $stats$nSelf
#> [1] 1910
#> 
#> $stats$nDumped
#> [1] 32
#> 
#> $stats$nFiltered
#> [1] 53553
#> 
#> $stats$nDups
#> [1] 613
#> 
#> $stats$nUnique
#> [1] 52940
#> 
#> $stats$threshold_uncut
#> [1] 7
#> 
#> $stats$threshold_self
#> [1] 7

2 Optional parameters

Extra optional arguments can be passed to the hicstuff workhorse library:

• iterative (default: TRUE): By default, hicstuff first truncates your set of reads to 20bp and attempts to align the truncated reads, then moves on to aligning 40bp-truncated reads for those which could not be mapped, etc. This procedure is longer than a traditional mapping but allows for more pairs to be rescued. Set to FALSE if you want to perform standard alignment of fastq files without iterative alignment;
• balancing_args (default: " --min-nnz 10 --mad-max 5 "): Specify here any balancing argument to be used by cooler when normalizing the binned contact matrices. Full list of options available at cooler documentation website;
• threads (default: 1L): Number of CPUs to use to process data;
• exclude_chr (default: 'Mito|chrM|MT'): List here any chromosome you wish to remove from the final contact matrix file;
• keep_bam (default: FALSE): Set to TRUE if you wish to keep the pair of .bam files;
• scratch (default: tempdir()): Points to a temporary directory to be used for processing.

3 Output files

The important files generated by HiCool are the following:

• A log file: <output_folder>/logs/<prefix>^mapped-<genome>^<hash>.log
• A multi-resolution, balanced contact matrix file: <output_folder>/matrices/<prefix>^mapped-<genome>^<hash>.mcool
• A .pairs file: <output_folder>/pairs/<prefix>^mapped-<genome>^<hash>.pairs
• Several diagnosis plots: <output_folder>/plots/<prefix>^mapped-<genome>^<hash>_*.pdf.

The diagnosis plots illustrate how pairs were filtered during the processing, using a strategy described in Cournac et al., BMC Genomics 2012. The event_distance chart represents the frequency of ++, +-, -+ and -- pairs in the library, as a function of the number of restriction sites between each end of the pairs, and shows the inferred filtering threshold. The event_distribution chart indicates the proportion of each type of pairs (e.g. dangling, uncut, abnormal, …) and the total number of pairs retained (3D intra + 3D inter).

Notes:

• .pairs file format is defined by the 4DN consortium;
• .(m)cool file format is defined by cooler authors in the supporting publication.

4 System dependencies

Processing Hi-C sequencing libraries into .pairs and .mcool files requires several dependencies, to (1) align reads to a reference genome, (2) manage alignment files (SAM), (3) filter pairs, (4) bin them to a specific resolution and (5)

All system dependencies are internally managed by basilisk.utils. HiCool maintains a conda environment containing:

• python 3.12.11
• numpy 1.26.4
• pandas 2.3.3
• bowtie2 2.5.4
• chromosight 1.6.3
• cooler 0.10.4
• hicstuff 3.2.5
• pairtools 1.1.3
• samtools 1.22.1

The first time HiCool() is executed, a fresh conda environment will be created and required dependencies automatically installed. This ensures compatibility between the different system dependencies needed to process Hi-C fastq files. The tools of this environment run in a separate process, so their libraries never conflict with those already loaded in the R session.

5 Session info

sessionInfo()
#> R version 4.6.1 (2026-06-24)
#> Platform: x86_64-pc-linux-gnu
#> Running under: Ubuntu 24.04.5 LTS
#> 
#> Matrix products: default
#> BLAS:   /home/biocbuild/bbs-3.24-bioc/R/lib/libRblas.so 
#> LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.12.0  LAPACK version 3.12.0
#> 
#> locale:
#>  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
#>  [3] LC_TIME=en_GB              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: America/New_York
#> tzcode source: system (glibc)
#> 
#> attached base packages:
#> [1] stats     graphics  grDevices utils     datasets  methods   base     
#> 
#> other attached packages:
#>  [1] HiContactsData_1.15.0 ExperimentHub_3.3.2   AnnotationHub_4.3.2  
#>  [4] BiocFileCache_3.3.0   dbplyr_2.6.0          BiocGenerics_0.59.12 
#>  [7] generics_0.1.4        HiCool_1.13.2         HiCExperiment_1.13.1 
#> [10] BiocStyle_2.41.0     
#> 
#> loaded via a namespace (and not attached):
#>  [1] tidyselect_1.2.1            viridisLite_0.4.3          
#>  [3] blob_1.3.0                  dplyr_1.2.1                
#>  [5] farver_2.1.2                Biostrings_2.81.9          
#>  [7] filelock_1.0.3              S7_0.2.2                   
#>  [9] fastmap_1.2.0               digest_0.6.39              
#> [11] lifecycle_1.0.5             KEGGREST_1.53.6            
#> [13] RSQLite_3.53.3              magrittr_2.0.5             
#> [15] compiler_4.6.1              rlang_1.3.0                
#> [17] sass_0.4.10                 tools_4.6.1                
#> [19] yaml_2.3.12                 data.table_1.18.6.1        
#> [21] knitr_1.52                  S4Arrays_1.13.2            
#> [23] htmlwidgets_1.6.4           curl_8.0.0                 
#> [25] bit_4.6.0                   DelayedArray_0.39.8        
#> [27] RColorBrewer_1.1-3          abind_1.4-8                
#> [29] BiocParallel_1.47.0         withr_3.0.3                
#> [31] purrr_1.2.2                 grid_4.6.1                 
#> [33] stats4_4.6.1                Rhdf5lib_2.1.0             
#> [35] ggplot2_4.0.3               scales_1.4.0               
#> [37] dichromat_2.0-1             SummarizedExperiment_1.43.0
#> [39] cli_3.6.6                   rmarkdown_2.32             
#> [41] crayon_1.5.3                otel_0.2.0                 
#> [43] httr_1.4.9                  tzdb_0.5.0                 
#> [45] sessioninfo_1.2.4           BiocBaseUtils_1.15.1       
#> [47] DBI_1.3.0                   cachem_1.1.0               
#> [49] rhdf5_2.57.19               parallel_4.6.1             
#> [51] AnnotationDbi_1.75.2        BiocManager_1.30.27        
#> [53] XVector_0.53.0              matrixStats_1.5.0          
#> [55] rmdformats_1.0.4            vctrs_0.7.3                
#> [57] Matrix_1.7-6                jsonlite_2.0.0             
#> [59] dir.expiry_1.21.0           bookdown_0.48              
#> [61] IRanges_2.47.5              S4Vectors_0.51.10          
#> [63] bit64_4.8.6                 crosstalk_1.2.2            
#> [65] plotly_4.12.1               strawr_0.0.92              
#> [67] jquerylib_0.1.4             tidyr_1.3.2                
#> [69] glue_1.8.1                  codetools_0.2-20           
#> [71] gtable_0.3.6                BiocVersion_3.24.0         
#> [73] BiocIO_1.23.3               GenomicRanges_1.65.4       
#> [75] tibble_3.3.1                pillar_1.11.1              
#> [77] rappdirs_0.3.4              basilisk.utils_1.25.0      
#> [79] htmltools_0.5.9             Seqinfo_1.3.2              
#> [81] rhdf5filters_1.25.4         httr2_1.3.0                
#> [83] R6_2.6.1                    vroom_1.7.1                
#> [85] evaluate_1.0.5              lattice_0.23-1             
#> [87] Biobase_2.73.2              png_0.1-9                  
#> [89] memoise_2.0.1               bslib_0.12.0               
#> [91] Rcpp_1.1.2                  InteractionSet_1.41.0      
#> [93] SparseArray_1.13.4          xfun_0.61                  
#> [95] MatrixGenerics_1.25.0       pkgconfig_2.0.3