Developed by:
Alberto Riva
DiBiG – ICBR Bioinformatics
University of Florida

CSCALL is a command-line tool for the analysis of sodium-bisulfite sequencing data for genome-wide DNA methylation studies. It provides functions to create an index of methylation sites in a reference genome (in arbitrary contexts), to call methylation at these sites, to perform quality-control on the input reads, and to generate alignments of bisulfite converted reads for clustering.


cscall is designed to be included in a comprehensive methylation analysis pipeline. The following is an example of how to set up such pipeline:

  1. Use cscall -build to create an index of the reference genome for the methylation site(s) being studied. This index only needs to be created once for each reference genome / methylation site.
  2. If necessary, use cscall -filter to perform quality control on the input reads. This function discards reads showing evidence of incomplete sodium bisulfite conversion.
  3. Align input reads to the reference genome. This step can be performed by a bisulfite-conversion-aware aligner such as bsmap. The output of this step will normally be a BAM file.
  4. Use cscall -call to call methylation rates at conversion sites in one or more samples. This step takes as input the BAM file(s) produced by the aligner, and produces one or more BED files.
  5. Optionally, use cscall -align on the BAM file(s) to generate alignments of reads in high-coverage regions. The alignments can be used as input to clustering programs, for example to detect the presence of subpopulations of cells with different methylation patterns.
  6. Evaluate differential methylation using e.g. the mcomp tool from the MOABS pipeline.


cscall is distributed as a GNU/Linux command-line 64bit executable. Source code is available upon request.

Download cscall

Cscall is also available on HiPerGator, in the dibig_tools module. To use it, execute:

module load dibig_tools


cscall provides an integrated help system. Please use

cscall -help Commands

to get a description of all cscall commands,

cscall -help all

to get a description of all command-line arguments, or

cscall -help option

to get a detailed description of an individual option.