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 environment.

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

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

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

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', 
    resolutions = c(4000, 8000, 16000), 
    genome = 'R64-1-1', 
    output = './HiCool/'
)
#> 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...
#> + /var/cache/basilisk/1.16.0/0/bin/conda create --yes --prefix /var/cache/basilisk/1.16.0/HiCool/1.4.0/env 'python=3.7.12' --quiet -c conda-forge -c bioconda
#> + /var/cache/basilisk/1.16.0/0/bin/conda install --yes --prefix /var/cache/basilisk/1.16.0/HiCool/1.4.0/env 'python=3.7.12' -c conda-forge -c bioconda
#> + /var/cache/basilisk/1.16.0/0/bin/conda install --yes --prefix /var/cache/basilisk/1.16.0/HiCool/1.4.0/env -c conda-forge -c bioconda 'python=3.7.12' 'python=3.7.12' 'bowtie2=2.5.0' 'samtools=1.16.1' 'hicstuff=3.1.5' 'chromosight=1.6.3' 'cooler=0.9.1'
#> HiCool :: Initiating processing of fastq files [tmp folder: /tmp/Rtmp1IvRUI/D3PESI]...
#> HiCool :: Mapping fastq files...
#> HiCool :: Removing unwanted chromosomes...
#> HiCool :: Parsing pairs into .cool file...
#> HiCool :: Generating multi-resolution .mcool file...
#> HiCool :: Balancing .mcool file...
#> 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/RtmpO3mTwa/Rbuild33040d5f8e4954/HiCool/vignettes/HiCool/1b2499c74cd3c_7833^mapped-R64-1-1^D3PESI.html
#> HiCool :: All processing successfully achieved. Congrats!
hcf
#> CoolFile object
#> .mcool file: ./HiCool//matrices/1b2499c74cd3c_7833^mapped-R64-1-1^D3PESI.mcool 
#> resolution: 4000 
#> pairs file: ./HiCool//pairs/1b2499c74cd3c_7833^mapped-R64-1-1^D3PESI.pairs 
#> metadata(3): log args stats
S4Vectors::metadata(hcf)
#> $log
#> [1] "./HiCool//logs/1b2499c74cd3c_7833^mapped-R64-1-1^D3PESI.log"
#> 
#> $args
#> $args$r1
#> [1] "/home/biocbuild/.cache/R/ExperimentHub/1b2499c74cd3c_7833"
#> 
#> $args$r2
#> [1] "/home/biocbuild/.cache/R/ExperimentHub/1b24993a81272d_7834"
#> 
#> $args$genome
#> [1] "/tmp/Rtmp1IvRUI/R64-1-1"
#> 
#> $args$resolutions
#> [1] "4000"
#> 
#> $args$resolutions
#> [1] "8000"
#> 
#> $args$resolutions
#> [1] "16000"
#> 
#> $args$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/Rtmp1IvRUI"
#> 
#> $args$wd
#> [1] "/tmp/RtmpO3mTwa/Rbuild33040d5f8e4954/HiCool/vignettes"
#> 
#> 
#> $stats
#> $stats$nFragments
#> [1] 1e+05
#> 
#> $stats$nPairs
#> [1] 73993
#> 
#> $stats$nDangling
#> [1] 10027
#> 
#> $stats$nSelf
#> [1] 2205
#> 
#> $stats$nDumped
#> [1] 83
#> 
#> $stats$nFiltered
#> [1] 61678
#> 
#> $stats$nDups
#> [1] 719
#> 
#> $stats$nUnique
#> [1] 60959
#> 
#> $stats$threshold_uncut
#> [1] 7
#> 
#> $stats$threshold_self
#> [1] 7

2 Optional parameters

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

3 Output files

The important files generated by HiCool are the following:

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:

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. HiCool maintains a basilisk environment containing:

The first time HiCool() is executed, a fresh basilisk environment will be created and required dependencies automatically installed. This ensures compatibility between the different system dependencies needed to process Hi-C fastq files.

5 Session info

sessionInfo()
#> R version 4.4.0 beta (2024-04-15 r86425)
#> Platform: x86_64-pc-linux-gnu
#> Running under: Ubuntu 22.04.4 LTS
#> 
#> Matrix products: default
#> BLAS:   /home/biocbuild/bbs-3.19-bioc/R/lib/libRblas.so 
#> LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.10.0
#> 
#> locale:
#>  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
#>  [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    
#>  [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_1.5.3 ExperimentHub_2.12.0 AnnotationHub_3.12.0
#> [4] BiocFileCache_2.12.0 dbplyr_2.5.0         BiocGenerics_0.50.0 
#> [7] HiCool_1.4.0         HiCExperiment_1.4.0  BiocStyle_2.32.0    
#> 
#> loaded via a namespace (and not attached):
#>   [1] DBI_1.2.2                   rlang_1.1.3                
#>   [3] magrittr_2.0.3              matrixStats_1.3.0          
#>   [5] compiler_4.4.0              RSQLite_2.3.6              
#>   [7] dir.expiry_1.12.0           png_0.1-8                  
#>   [9] vctrs_0.6.5                 stringr_1.5.1              
#>  [11] pkgconfig_2.0.3             crayon_1.5.2               
#>  [13] fastmap_1.1.1               XVector_0.44.0             
#>  [15] rmdformats_1.0.4            utf8_1.2.4                 
#>  [17] rmarkdown_2.26              sessioninfo_1.2.2          
#>  [19] tzdb_0.4.0                  UCSC.utils_1.0.0           
#>  [21] strawr_0.0.91               purrr_1.0.2                
#>  [23] bit_4.0.5                   xfun_0.43                  
#>  [25] zlibbioc_1.50.0             cachem_1.0.8               
#>  [27] GenomeInfoDb_1.40.0         jsonlite_1.8.8             
#>  [29] blob_1.2.4                  rhdf5filters_1.16.0        
#>  [31] DelayedArray_0.30.0         Rhdf5lib_1.26.0            
#>  [33] BiocParallel_1.38.0         parallel_4.4.0             
#>  [35] R6_2.5.1                    bslib_0.7.0                
#>  [37] stringi_1.8.3               reticulate_1.36.1          
#>  [39] GenomicRanges_1.56.0        jquerylib_0.1.4            
#>  [41] Rcpp_1.0.12                 bookdown_0.39              
#>  [43] SummarizedExperiment_1.34.0 knitr_1.46                 
#>  [45] IRanges_2.38.0              Matrix_1.7-0               
#>  [47] tidyselect_1.2.1            abind_1.4-5                
#>  [49] yaml_2.3.8                  codetools_0.2-20           
#>  [51] curl_5.2.1                  lattice_0.22-6             
#>  [53] tibble_3.2.1                withr_3.0.0                
#>  [55] KEGGREST_1.44.0             InteractionSet_1.32.0      
#>  [57] Biobase_2.64.0              basilisk.utils_1.16.0      
#>  [59] evaluate_0.23               Biostrings_2.72.0          
#>  [61] pillar_1.9.0                BiocManager_1.30.22        
#>  [63] filelock_1.0.3              MatrixGenerics_1.16.0      
#>  [65] stats4_4.4.0                plotly_4.10.4              
#>  [67] generics_0.1.3              vroom_1.6.5                
#>  [69] BiocVersion_3.19.1          S4Vectors_0.42.0           
#>  [71] ggplot2_3.5.1               munsell_0.5.1              
#>  [73] scales_1.3.0                glue_1.7.0                 
#>  [75] lazyeval_0.2.2              tools_4.4.0                
#>  [77] BiocIO_1.14.0               data.table_1.15.4          
#>  [79] rhdf5_2.48.0                grid_4.4.0                 
#>  [81] tidyr_1.3.1                 crosstalk_1.2.1            
#>  [83] AnnotationDbi_1.66.0        colorspace_2.1-0           
#>  [85] GenomeInfoDbData_1.2.12     basilisk_1.16.0            
#>  [87] cli_3.6.2                   rappdirs_0.3.3             
#>  [89] fansi_1.0.6                 S4Arrays_1.4.0             
#>  [91] viridisLite_0.4.2           dplyr_1.1.4                
#>  [93] gtable_0.3.5                sass_0.4.9                 
#>  [95] digest_0.6.35               SparseArray_1.4.0          
#>  [97] htmlwidgets_1.6.4           memoise_2.0.1              
#>  [99] htmltools_0.5.8.1           lifecycle_1.0.4            
#> [101] httr_1.4.7                  mime_0.12                  
#> [103] bit64_4.0.5