11. IHEC standard analyses

To perform a standard IHEC WGBS analysis it is necessary to have an up-to-date version of gemBS installed, and to set the analysis parameters appropriately. To help with the configuration issue, gemBS includes standard configuration files that can be included in the parameter configuration files to ensure that the standard pipeline behaviour is achieved.

The gemBS distribution includes three IHEC analysis configuration files:

IHEC_standard.conf
IHEC_RRBS.conf
IHEC_PBAT.conf

An example configuration file that uses IHEC_standard.conf can be seen here: Example configuration file.

Note

The position of the include statement in the configuration file is important: variables must be included before they are referenced, and new variable definitions will overwrite existing definitions.

Once the parameter files have been set up, then running the standard set of gemBS commands (map, call, extract, map-report and call-report) will perform the standard pipeline and generate the required outputs.

11.1. IHEC standard configuration files

We can see the contents of the three files below:

  • IHEC_standard.conf

[index]

sampling_rate = 4

[mapping]

non_stranded = False
remove_individual_bams = True

[calling]

mapq_threshold = 10
qual_threshold = 13
reference_bias = 2
left_trim = 5
right_trim = 0
keep_improper_pairs = False
keep_duplicates = False
haploid = False
conversion = auto
remove_individual_bcfs = True

# Contigs smaller than contig_pool_limit will be called together
contig_pool_limit = 25000000

[extract]

strand_specific = True
phred_threshold = 10
make_cpg = True
make_non_cpg = True
make_bedmethyl = True
make_bigwig = True
  • IHEC_RRBS.conf

include IHEC_standard.conf

[calling]

keep_duplicates = True
  • IHEC_PBAT.conf

include IHEC_standard.conf

[mapping]

non-stranded = True

[calling]

keep_improper_pairs = True

We can see that the IHEC_RRBS.conf and IHEC_PBAT.conf configuration files are simply slight modifications of the IHEC_standard.conf file, obtained by including the standard file and then overwriting key parameters.

11.2 Use of gemBS containers

To ensure that gemBS is installed correctly and is up-to-date, it can be convenient to make use of containerized versions of gemBS (Singularity or Docker). This simplifies the installation process and makes sure that the support libraries and applications needed by gemBS are installed correctly and are the right versions (see 10. Containers for more details).