hicstuff_usage.md

Usage

This document present the usage and parameters of the hicstuff workflow. To see the parameters of the hicpro workflow, go here. Samplesheet input and core arguments are detailed there.

Parameters

Inputs

Inputs are the same as the hicpro workflow

--input

Use this to specify the location of your input FastQ files. For example:

--input 'path/to/data/sample_*_{1,2}.fastq'

Please note the following requirements:

1. The path must be enclosed in quotes
2. The path must have at least one * wildcard character
3. When using the pipeline with paired end data, the path must use {1,2} notation to specify read pairs.

If left unspecified, a default pattern is used: data/*{1,2}.fastq.gz

Note that the Hi-C data analysis workflow requires paired-end data.

Reference genomes

The pipeline config files come bundled with paths to the Illumina iGenomes reference index files. If running with docker or AWS, the configuration is set up to use the AWS-iGenomes resource.

--genome (using iGenomes)

There are many different species supported in the iGenomes references. To run the pipeline, you must specify which to use with the --genome flag.

You can find the keys to specify the genomes in the iGenomes config file.

--fasta

If you prefer, you can specify the full path to your reference genome when you run the pipeline:

--fasta '[path to Fasta reference]'

--bwt2_index

The bowtie2 indexes are required to align the data with the HiC-Pro workflow. If the --bwt2_index is not specified, the pipeline will either use the iGenomes bowtie2 indexes (see --genome option) or build the indexes on-the-fly (see --fasta option)

--bwt2_index '[path to bowtie2 index]'

--chromosome_size

The Hi-C pipeline also requires a two-column text file with the chromosome name and the chromosome size (tab-separated). If not specified, this file will be automatically created by the pipeline. In the latter case, the --fasta reference genome has to be specified.

   chr1    249250621
   chr2    243199373
   chr3    198022430
   chr4    191154276
   chr5    180915260
   chr6    171115067
   chr7    159138663
   chr8    146364022
   chr9    141213431
   chr10   135534747
   (...)

--chromosome_size '[path to chromosome size file]'

--restriction_fragments

Finally, Hi-C experiments based on restriction enzyme digestion require a BED file with coordinates of restriction fragments.

   chr1   0       16007   HIC_chr1_1    0   +
   chr1   16007   24571   HIC_chr1_2    0   +
   chr1   24571   27981   HIC_chr1_3    0   +
   chr1   27981   30429   HIC_chr1_4    0   +
   chr1   30429   32153   HIC_chr1_5    0   +
   chr1   32153   32774   HIC_chr1_6    0   +
   chr1   32774   37752   HIC_chr1_7    0   +
   chr1   37752   38369   HIC_chr1_8    0   +
   chr1   38369   38791   HIC_chr1_9    0   +
   chr1   38791   39255   HIC_chr1_10   0   +
   (...)

If not specified, this file will be automatically created by the pipeline. In this case, the --fasta reference genome will be used. Note that the --digestion or --restriction_site parameter is mandatory to create this file.

Digestion of the reads

--cutsite

Generates new gzipped fastq files from original fastq. The function will cut the reads at their religation sites and creates new pairs of reads with the different fragments obtained after cutting at the digestion sites. Default: false

--cutsite

⚠️ Warning: This option cannot be used at the same time as the iterative mapping.

--cutsite_mode

Mode to use to make the digestion. Three values possible: "all", "for_vs_rev", "pile". Default: for_vs_rev

--cutsite_mode '[all | for_vs_rev | pile]'

--cutsite_seed

Minimum size of a fragment (i.e. seed size used in mapping as reads smaller won't be mapped.) Default: 0

--cutsite_seed '[Size of fragment]'

Hicstuff specific parameters

The following options are defined in the nextflow.config file, and can be updated either using a custom configuration file (see -c option) or using command line parameters.

Mapping

--hicstuff_bwt2_align_opts

Bowtie2 alignment option for normal mode end-to-end mapping. Default: '--very-sensitive-local'

--hicstuff_bwt2_align_opts '[Options for bowtie2 mapping on full reads]'

--iteralign

Truncate reads from a fastq file to 20 basepairs and iteratively extend and re-align the unmapped reads to optimize the proportion of uniquely aligned reads in a 3C library. Default: false

--iteralign

Use --hicstuff_min_qual to set the minimum quality for the iterative alignment.

--hicstuff_read_len

Read length in the fasta file. If set to None, the length of the first read is used. Set this value to the longest read length in the file if you have different read lengths. Default: None

--hicstuff_read_len '[Wanted read length]'

--save_bam

If specified, save BAM files after mapping (works for normal and iterative mode). Default: false

--save_bam

Fragment enzyme

--hicstuff_min_size

Minimum contig size required to keep it. Default:0

--hicstuff_min_size '[Minimum size value]'

--hicstuff_circular

Use if the genome is circular. Default:false

--hicstuff_circular

--hicstuff_output_contigs

Name of info contigs file. Default: 'info_contigs.txt'

--hicstuff_output_contigs '[Name of info contigs file]'

--hicstuff_output_frags

Name of fragments list file. Default: 'fragments_list.txt'