Specialized Functions Walkthrough

We cover the most high level use cases in our general walkthrough. However, there are some functions available in methylprep for less common (more specialized) use cases that we’ll cover here.

Building a composite dataset with alert and composite

methylprep includes a few functions that can be used in a pipeline to merge datasets from GEO into one larger dataset. Say, for example, that you are particularly interested in investigating data from glioblastoma (an aggressive form of cancer that can occur in the brain or spine).

You may use the alert command to compile a csv of GEO datasets that mention the keyword glioblastoma. The csv will contain, at most, 500 rows (a query limit set by GEO).

>>> python -m methylprep alert -k "glioblastoma"

After looking through the .csv file and eliminating any datasets that aren’t useful, you can pull the column of GSE id’s and run methylprep composite from the CLI. composite will run the download command in a loop given a text file with a list of GSE id’s.

From our geo alert glioblastoma .csv file, here are 4 datasets that we might pull into one larger composite dataset. One thing to be aware of is to make sure that the platform types are all the same. Otherwise you may be combining 450k array data with EPIC array data.

Here is a peek at the txt file with the list of GSEs. Make sure that the format is such that there is just one GSE id per line.

>>> cat glioblastoma.txt

Now we can run the composite command and it’ll download/process data from each GSE in the list. You need to use -l to specify the .txt file that contains the GSEs and -d to specify the directory where to save the data.

>>> python -m methylprep composite -l ~/tutorial/glioblastoma.txt -d ~/tutorial

The meta_data function has a similar application for building a composite dataset when using the --control option. See below for the walkthrough of how to use that command to build a dataset of healthy control samples instead of experimental/disease case samples.

using the meta_data function

Creating a meta_data.pkl file

GEO data will always be accompanied by a MINiML file (an .xml file that contains information about the platform, series, and samples). methylprep will handle downloading and parsing this as part of the download or process functions, with the output stored in the meta_data.pkl file. So if you’re going to run either of those, you don’t need to worry about running meta_data separately.

However, if you are interested in creating a meta_data.pkl file without downloading/processing data, the meta_data function will come in handy.

>>> python -m methylprep meta_data -i GSE147391 -d ~/tutorial
INFO:methylprep.download.miniml:found 16 idat files; updated meta_data.
INFO:methylprep.download.miniml:Final samplesheet contains 16 rows and 9 columns

Filtering samples with meta_data

meta_data also has a few experimental commands that may be useful in filtering unwanted samples and reducing the time needed to process datasets. For example, consider the glioma dataset from GSE147391 (the dataset discussed in the general walkthrough). This dataset contains methylation profiles of high and low grade gliomas from 16 patients (8 high grade gliomas and 8 low grade gliomas). If we wanted to ignore the high grade gliomas and focus on the low grade gliomas, we could use meta_data’s --keyword (-k for short) command to search for samples that are indicated as “grade II” in the sample sheet and only retain those values.

>>> python -m methylprep meta_data -i GSE147391 -d ~/tutorial -k "grade II"
INFO:methylprep.download.miniml:found 16 idat files; updated meta_data.
INFO:methylprep.download.miniml:Filtering keyword: Retained 8/16 samples matching `grade II``.
INFO:methylprep.download.miniml:Final samplesheet contains 8 rows and 9 columns

Now our meta_data.pkl file only contains information on the low grade gliomas, and we can exclude the other samples from our future analyses. If we haven’t already used methylprep process on our data, we can also include the --sample_name or -n flag with the list of the samples we want to run (the low grade gliomas) to save time processing the data.

building a composite dataset using meta_data

To better demonstrate the use case for meta_data's --control command, we will work with a new dataset. GSE163970 is a dataset that examines differential methylation in 253 patients with Paget’s disease of bone (PDB) as compared to 280 controls.

We might be able to cut down processing time for this large dataset if we are just interested in the control data and want to exclude the PDB samples. One example of a scenario in which this would be helpful is building a large composite dataset that includes only healthy control samples from multiple GEO datasets.

Before downloading any data from GEO, we’ll run this:

>>> python -m methylprep meta_data --control -i GSE163970

methylprep will look through our computer and realize that we don’t have any data or metadata files downloaded. It will then pull the MINiML file from the GEO database and search it for samples with the word “control” or “ctrl” in the metadata.

INFO:methylprep.download.miniml:Downloading GSE163970_family.xml.tgz
INFO:methylprep.download.miniml:Filtering controls: Retained 260/492 samples matching ['control', 'ctrl'].
INFO:methylprep.download.miniml:Final samplesheet contains 260 rows and 13 columns

As we noted above, these functions may not work on every dataset. methylprep does its best to identify controls based on keywords like ‘ctrl’ or ‘control’, but controls may be labelled differently in various datasets.

using beta_bake for preprocessed data

beta_bake is a function intended for users who may want to build a composite dataset with mixed data formats. Say we ran the alert command and found 2 candidate datasets that we are interested in combining into a larger dataset: GSE164149 and GSE158089. The problem? GSE158089 doesn’t have IDAT files to download!

Using beta_bake, we can download the available data from GEO (whether it is raw IDAT files or has already been processed into beta values).

GSE164149 is a dataset of 16 samples run on EPIC arrays. This data shows the methylome of CRISPR-mediated gene knock-ins on Tregs with high expansion (the researchers chose FOXP3 as the target benchmark because it is a master transcription factor). This dataset includes pre-processed beta values in the GEO data, but beta_bake will preferentially pull the raw IDATs (also included) so that we are able to process them with methylprep.

The command will look like this:

>>> python -m methylprep beta_bake -i GSE164149 -d ~/tutorial/GSE164149

The -d option specifies which directory to store the downloaded files in.

The output files in this case include:

  • geo_alert GSE164149.csv: a csv file of the results of running the methylprep alert command with the keyword “GSE164149”
  • GSE164149_family.xml: the MINiML file that contains sample metadata.
  • GSE164149_GPL21145_meta_data.pkl: a python-optimized .pkl file that holds the same metadata found in the MINiML file.
  • GSE164149_GPL21145_samplesheet.csv: the sample sheet for this dataset.
  • 32 .idat files: the raw IDAT data for our 16 samples.
  • GSE164149.zip: a compressed folder that holds of all of the files above.

After downloading these files, we run methylprep process in this folder, and we will have a beta_values.pkl file to use for analysis:

>>> python -m methylprep process --all -d ~/tutorial/GSE164149

The second dataset, GSE158089, is a set of 14 samples that investigates DNA methylomic trajectories of neuronal aging. They sampled induced pluripotent stem cell lines at 4 different points during development and ran those samples on the EPIC array. This dataset only includes the processed beta values, so beta_bake will just pull those into a .pkl format for us.

>>> python -m methylprep beta_bake -i GSE158089 -d ~/tutorial/GSE158089

The output files we have for this are:

  • GSE158089_beta_values.pkl.gz: beta values stored in a compressed .pkl file (run gunzip from the command line to unzip this, or just double click on a Mac).
  • GSE158089_GPL21145_meta_data.pkl.gz: a similarly formatted file that holds the meta_data found for these samples.

Now that we have two beta_values.pkl files for two datasets from the same array types, we can load these into an IDE and easily combine them, the same way you would combine any two pandas dataframes.

import pandas as pd
import methylcheck

GSE158089_betas = pd.read_pickle('~/tutorial/GSE158089/GSE158089_beta_values.pkl')

GSE164149_betas = pd.read_pickle('~/tutorial/GSE164149/beta_values.pkl')

betas = pd.concat([GSE158089_betas, GSE164149_betas], axis=1)