2. Preparing Files¶

The pipeline is controlled through editing configuration and manifest files. Defaults are found in the /WORKDIR/config and /WORKDIR/manifest directories, after initialization.

2.1 Configs¶

The configuration files control parameters and software of the pipeline. These files are listed below:

• config/config.yaml
• resources/cluster.yaml
• resources/tools.yaml

2.1.1 Cluster Config¶

The cluster configuration file dictates the resouces to be used during submission to Biowulf HPC. There are two differnt ways to control these parameters - first, to control the default settings, and second, to create or edit individual rules. These parameters should be edited with caution, after significant testing.

2.1.2 Tools Config¶

The tools configuration file dictates the version of each software or program that is being used in the pipeline.

2.1.3 Config YAML¶

There are several groups of parameters that are editable for the user to control the various aspects of the pipeline. These are :

• Folders and Paths
• These parameters will include the input and ouput files of the pipeline, as well as list all manifest names.
• User parameters
• These parameters will control the pipeline features. These include thresholds and whether to perform processes.
• References
• These parameters will control the location of index files, spike-in references, adaptors and species calling information.

2.1.3.1 User Parameters¶

2.1.3.1.1 (Spike in Controls)¶

The pipeline allows for the use of a species specific spike-in control, or the use of normalization via library size. The parameter spikein_genome should be set to the species term used in spikein_reference.

For example for ecoli spike-in:

run_contrasts: true
norm_method: "spikein"
spikein_genome: "ecoli"
spikein_reference:
  ecoli:
    fa: "PIPELINE_HOME/resources/spikein/Ecoli_GCF_000005845.2_ASM584v2_genomic.fna"

For example for drosophila spike-in:

run_contrasts: true
norm_method: "spikein"
spikein_genome: "drosophila"
spikein_reference:
  drosophila:
    fa: "/fdb/igenomes/Drosophila_melanogaster/UCSC/dm6/Sequence/WholeGenomeFasta/genome.fa"

If it's determined that the amount of spike-in is not sufficient for the run, a library normaliaztion can be performed.

1. Complete a CARLISLE run with spike-in set to "Y". This will allow for the complete assessment of the spike-in.
2. Run inital QC analysis on the output data
3. Add the alignment_stats dir to the configuration file.
4. Re-run the CARLISLE pipeline

2.1.3.1.2 Duplication Status¶

Users can select duplicated peaks (dedup) or non-deduplicated peaks (no_dedup) through the user parameter.

dupstatus: "dedup, no_dedup"

2.1.3.1.3 Peak Caller¶

Three peak callers are available for deployment within the pipeline, with different settings deployed for each caller.

1. MACS2 is available with two peak calling options: narrowPeak or broadPeak. NOTE: DESeq step generally fails for broadPeak; generally has too many calls.

peaktype: "macs2_narrow, macs2_broad,"

1. SEACR is available with four peak calling options: stringent or relaxed parameters, to be paired with "norm" for samples without a spike-in control and "non" for samples with a spikein control

peaktype: "seacr_stringent, seacr_relaxed"

1. GOPEAKS is available with two peak calling options: narrowpeaks or broadpeaks

peaktype: "gopeaks_narrow, gopeaks_broad"

A complete list of the available peak calling parameters and the recommended list of parameters is provided below:

# Recommended list
### peaktype: "macs2_narrow, macs2_broad, gopeaks_narrow, gopeaks_broad"

# Available list
### peaktype: "macs2_narrow, macs2_broad, seacr_norm_stringent, seacr_norm_relaxed, seacr_non_stringent, seacr_non_relaxed, gopeaks_narrow, gopeaks_broad"

2.1.3.1.3.1 Macs2 additional option¶

MACS2 can be run with or without the control. adding a control will increase peak specificity Selecting "Y" for the macs2_control will run the paired control sample provided in the sample manifest

2.1.3.1.4 Quality Tresholds¶

Thresholds for quality can be controled through the quality_tresholds parameter. This must be a list of comma separated values. minimum of numeric value required.

• default MACS2 qvalue is 0.05 https://manpages.ubuntu.com/manpages/xenial/man1/macs2_callpeak.1.html
• default GOPEAKS pvalue is 0.05 https://github.com/maxsonBraunLab/gopeaks/blob/main/README.md
• default SEACR FDR threshold 1 https://github.com/FredHutch/SEACR/blob/master/README.md

#default values
quality_thresholds: "0.1, 0.05, 0.01"

2.1.3.2 References¶

Additional reference files may be added to the pipeline, if other species were to be used.

The absolute file paths which must be included are:

1. fa: "/path/to/species.fa"
2. blacklist: "/path/to/blacklistbed/species.bed"

The following information must be included:

1. regions: "list of regions to be included; IE chr1 chr2 chr3"
2. macs2_g: "macs2 genome shorthand; IE mm IE hs"

2.2 Preparing Manifests¶

There are two manifests, one which required for all pipeliens and one that is only required if running a differential analysis. These files describe information on the samples and desired contrasts. The paths of these files are defined in the snakemake_config.yaml file. These files are:

• samplemanifest
• contrasts

2.2.1 Samples Manifest (REQUIRED)¶

This manifest will include information to sample level information. It includes the following column headers:

• sampleName: the sample name WITHOUT replicate number (IE "SAMPLE")
• replicateNumber: the sample replicate number (IE "1")
• isControl: whether the sample should be identified as a control (IE "Y")
• controlName: the name of the control to use for this sample (IE "CONTROL")
• controlReplicateNumber: the replicate number of the control to use for this sample (IE "1")
• path_to_R1: the full path to R1 fastq file (IE "/path/to/sample1.R1.fastq")
• path_to_R2: the full path to R1 fastq file (IE "/path/to/sample2.R2.fastq")

An example sampleManifest file is shown below:

2.2.2 Contrast Manifest (OPTIONAL)¶

This manifest will include sample information to performed differential comparisons.

An example contrast file:

Note: you must have more than one sample per condition in order to perform differential analysis with DESeq2