Easy-450k

This program creates prepared projects to analyze Illumina 450k array DNA methylation data by changing a bunch of config files without using extensive programming.

It will do the following for you:

• Create a project structure
• Create a set of R scripts that will be able run the whole pipeline, from reading the data to running statistical routines, using GNU Make
• Generate a report with QC plots and project statistics

Available pipeline steps are:

1. Reading GenomeStudio output
2. Filtering samples for consecutive analysis
3. Filtering probes based on various criteria
4. Background correction
5. Color bias adjustment
6. Quantile normalization
7. Probe bias correction
8. BMIQ
9. Batch correction
10. Statistical analysis (linear regression or Mann-Whitney-Wilcoxon test)

For more info, please read this guide (here will be the guide).

Dependencies

python3 (Works with python3 only) PyYaml

Installation

git clone https://github.com/petr-volkov/easy-450k

Usage

1. Create a new project

    python3 ${path_to_cloned_repo}/easy-450k.py project_name
   

2. Edit configuration files

3. Copy or link your GenomeStudio output file to RawData/input_file.txt

4. Run 'make' in the project dir.

Illumina 450k configuration options

1. Configs/analysis_samples.txt

Includes a list of samples, line by line, to be included in the analysis.

2. Configs/probe_filtering.yaml

Allows to define criteria to filter probes from the analysis.

To set up filtering steps, put the required parameters in a .yaml file like this

filtering:
    mean_detection: 0.01
    rs_probes : true
    ch_probes : true
    snp_targets: 0.1
    cross_reactive: 49
    x_chr: true
    y_chr: false

filtering: - denotes filtering section of the config mean_detection: 0.01 - removes probes with mean Illumina detection pvalue > 0.01
rs_probes : true - removes probes with mean Illumina detection pvalue > 0.01
ch_probes : true - removes Illumina non-CpG 'ch' probes
snp_targets: 0.1 - Remove probes with SNPs in a target CpG with dbSNP MAF at least 0.1 !!!! USE WITH CAUTION - LEGACY MODE - NOT FULLY IMPLEMENTED !!!!
cross_reactive: 49 - Removes probes that cross-hybridize to different genomic location with at least 49 probes (possible values - 47, 48, 49, 50).
x_chr: true - Remove x chromosome probes
y_chr: false - Remove y chromosome probes

3. Configs/normalization.yaml

Allows to define criteria to normalize dataset Example:

background_correct: true
adjust_color_bias: false
quantile_normalize: true

4. Configs/bmiq.yaml

Allows to define criteria to perform BMIQ color bias adjustment

Available options (with examples):

seed: 100
nfit: 50000
n_cores: 30

seed - random generator seed, so that the BMIQ results are reproducible nfit - Number of probes of a given design to use for the fitting. n_cores - how many parallel processess will be used to run bmiq

You can additionally set any other parameters of BMIQ function.

5. Configs/combat.yaml

Allows to define criteria to perform Combat batch correction

Config example:

phenotype_file: Configs/phenotype_file.txt
batch_column: Batch_Column_Name
sample_names_column: ID
covariates:
categorical:
- CategoricalA_Column_Name
- CategoricalB_Column_Name
numeric:
- NumericA_Column_Name
- NumericB_Column_Name
- NumericC_Column_Name	

The program will load the file Configs/phenotype_file.txt. Phenotype file should be a tab separated text file, with phenotypes and other variables of interest as columns, and samples as raws. Sample_names_column should correspond to a subset of sample names of the original GenomeStudio file. It is used to match phenotypes to samples, so it is important for it to be correct.

6. Configs/analysis.yaml

There are currently 2 analysis options, wilcoxon test and linear regression model (more tests will be added in the future).

For wilcoxon test, make the config file that looks like this:

phenotype_file: Configs/phenotype_file.txt
type: wilcoxon
n_cores: 5
group: CaseControl_Column_Name
paired: false
exact: true

Column with CaseControl_Column_Name should contain only the following values: 1, 2, -1. Samples with value 1 will be tested against samples with value 2. Samples with value -1 are ignored. If paired is set up to true, pairs will be determined by the order of samples in both vectors.

For linear regression tests, use the following:

phenotype_file: Configs/phenotype_file.txt
type: lm
n_cores: 46
covariates:
numeric:
	- NumericA_Column_Name
	- NumericB_Column_Name
categorical:
	- CategoricalA_Column_Name
	- CategoricalB_Column_Name
	- CategoricalC_Column_Name

This is file can be the same as Configs/combat.yaml, but it is not always the case. Therefore, 2 perhaps very similar config files are needed, to avoid more complex logic.