Split, Filter, Normalize and Integrate Sequencing Data

A Snakemake 8 workflow to split, filter, normalize, integrate and select highly variable features of count matrices resulting from experiments with sequencing readout (e.g., RNA-seq, ATAC-seq, ChIP-seq, Methyl-seq, miRNA-seq, …) including confounding factor analyses and diagnostic visualizations documenting the respective data transformations. This often represents the first analysis after signal processing, critically influencing all downstream analyses.

[!NOTE]
This workflow adheres to the module specifications of MrBiomics, an effort to augment research by modularizing (biomedical) data science. For more details, instructions, and modules check out the project’s repository.

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[!IMPORTANT]
If you use this workflow in a publication, please don’t forget to give credit to the authors by citing it using this DOI 10.5281/zenodo.8144219.

🖋️ Authors

• Stephan Reichl
• Christoph Bock

💿 Software

This project wouldn’t be possible without the following software and their dependencies.

Software	Reference (DOI)
ComplexHeatmap	https://doi.org/10.1093/bioinformatics/btw313
CQN	https://doi.org/10.1093/biostatistics/kxr054
edgeR	https://doi.org/10.1093/bioinformatics/btp616
fastcluster	https://doi.org/10.18637/jss.v053.i09
ggplot2	https://ggplot2.tidyverse.org/
limma	https://doi.org/10.1093/nar/gkv007
matplotlib	https://doi.org/10.1109/MCSE.2007.55
pandas	https://doi.org/10.5281/zenodo.3509134
patchwork	https://CRAN.R-project.org/package=patchwork
reComBat	https://doi.org/10.1093/bioadv/vbac071
reshape2	https://doi.org/10.18637/jss.v021.i12
scikit-learn	http://jmlr.org/papers/v12/pedregosa11a.html
seaborn	https://doi.org/10.21105/joss.03021
Snakemake	https://doi.org/10.12688/f1000research.29032.2
statsmodels	https://www.statsmodels.org/stable/index.html#citation
TMM	https://doi.org/10.1186/gb-2010-11-3-r25

🔬 Methods

This is a template for the Methods section of a scientific publication and is intended to serve as a starting point. Only retain paragraphs relevant to your analysis. References [ref] to the respective publications are curated in the software table above. Versions (ver) have to be read out from the respective conda environment specifications (workflow/envs/*.yaml file) or post execution in the result directory (spilterlize_integrate/envs/*.yaml). Parameters that have to be adapted depending on the data or workflow configurations are denoted in squared brackets e.g., [X].

Split. The input data was split by [split_by], with each split denoted by [split_by]_{annotation_level}. The complete data was retained in the “all” split. Sample filtering was achieved by removing sample rows from the annotation file or using NA in the respective annotation column. Annotations were also split and provided separately. The data was loaded, split, and saved using the Python library pandas (ver)[ref].

All downstream analyses were performed for each split separately.

Filter. The features were filtered using the filterByExpr function from the R package edgeR(ver)[ref]. The function was configured with the following parameters: group set to [group], min.count to [min.count], min.total.count to [min.total.count], large.n to [large.n], and min.prop to [min.prop]. The number of features was reduced from [X] to [X] by filtering.

Normalize. Normalization of the data was performed to correct for technical biases.

The CalcNormFactors function from the R package edgeR(ver)[ref] was used to normalize the data using the [edgeR_parameters.method] method with subsequent [edgeR_parameters.quantification] quantification. The parameters used for this normalization included [edgeR_parameters].

Conditional Quantile Normalization (CQN) was performed using the R package cqn(ver)[ref]. The parameters used for this normalization included [cqn_parameters].

The VOOM method from the R package limma(ver)[ref] was used to estimate the mean-variance relationship of the log-counts and generate a precision weight for each observation. The parameters used for this normalization included [voom_parameters].

The normalization results were log2-normalized for downstream analyses.

Integrate. The data integration was performed using the reComBat method(ver)[ref] and applied to the log-normalized data. This method adjusts for batch effects and unwanted sources of variation while retaining desired (e.g., biological) variability. The following effects were modeled within the integration: batch [batch_column], desired variation [desired_categorical] and [desired_numerical], unwanted variation [unwanted_categorical] and [unwanted_numerical]. The parameters used for the integration included [reComBat_parameters].

Highly Variable Feature (HVF) selection. Highly variable features (HVF) were selected based on the binned normalized dispersion of features adapted from Zheng (2017) Nature Communications. The top [hvf_parameters.top_percentage] percent of features were selected, resulting in [X] features. The dispersion for each feature across all samples was calculated as the standard deviation. Features were binned based on their means, and the dispersion of each feature was normalized by subtracting the median dispersion of its bin and dividing by the median absolute deviation (MAD) of its bin using the Python package statsmodels (ver)[ref]. The number of bins used for dispersion normalization was [hvf_parameters.bins_n]. The selected HVFs were visualized by histograms before and after normalization, mean to normalized dispersion scatterplots, and a scatterplot of the ranked normalized dispersion, always highlighting the selected features.

Confounding Factor Analysis (CFA). We assessed the potential confounding effects of metadata on principal components (PCs) by quantifying their statistical associations with the first ten PCs from principal component analysis (PCA). Categorical metadata were tested using the Kruskal-Wallis test, while numeric metadata were analyzed using Kendall’s Tau correlation. Metadata without variation were excluded, and numeric metadata with fewer than 25 unique values were converted to factors. P-values for associations between PCs and metadata were calculated and adjusted for multiple testing using the Benjamini-Hochberg method. The results were visualized as a heatmap with hierarchically clustered rows (metadata) displaying -log10 adjusted p-values, distinguishing between numeric and categorical metadata.

Correlation Heatmaps. To assess sample similarities we generated heatmaps of the sample-wise Pearson correlation matrix. using the cor function in R with the “pearson” method. Two versions of heatmaps were created: one hierarchically clustered and one sorted alphabetically by sample name. Hierarchical clustering was performed with the hclust function from the fastcluster package [ver] using “euclidean” distance and “complete” linkage. Heatmaps were visualized using the ComplexHeatmap package [ver] and annotated with relevant metadata.

Visualization. The quality of the data and the effectiveness of the processing steps were assessed through the following visualizations (raw/filtered counts were log2(x+1)-normalized): the mean-variance relationship of all features, densities of log2-values per sample, boxplots of log2-values per sample, and Principal Component Analysis (PCA) plots. For the PCA plots, features with zero variance were removed beforehand and colored by [visualization_parameters.annotate]. The plots were generated using the R libraries ggplot2, reshape2, and patchwork(ver)[ref].

The analyses and visualizations described here were performed using a publicly available Snakemake [ver](ref) workflow (ver) 10.5281/zenodo.8144219.

🚀 Features

The workflow performs the following steps to produce the outlined results:

• Split ({annotation_column}_{annotation_level}/counts.csv)
  • The input data is split according to the levels of the provided annotation column(s), and all downstream steps are performed for each split separately.
  • Each split is denoted by {annotation_column}_{annotation_level}.
  • The complete input data is retained in the split all.
  • Note: splits are performed solely based on the provided annotations, arbitrarily complex splits are possible as long as they are reflected in the annotations.
  • Sample filtering (e.g., QC) can be achieved within…
    • …all by removing the sample rows from the annotation file.
    • …splits by using NA in the respective annotation column.
  • Annotations are also split and provided separately ({annotation_column}_{annotation_level}/annotation.csv).
• Filter (filtered.csv)
  • The features are filtered using the edgeR package’s filterByExpr function, which removes low-count features that are unlikely to be informative but likely to be statistically problematic downstream.
  • The min.count parameter has the biggest impact on the filtering process, while min.total.count does not.
  • min.count is based on actual raw counts, not CPM (bioconductor).
  • The CPM cutoff is calculated as cpm_cutoff = min.count / medianLibSize * 1e6, using the median library size for normalization (biostars).
  • min.count.total operates purely only to raw counts, ignoring CPM or other normalization factors like library size, which is consistent with how raw counts are handled in filtering.
  • The desired number of features depends on the data and assay used, below are some examples that provide a ballpark estimate based on previous experiences (feel free to ignore).
    • Generally, you should filter until the mean-variance plot shows a consistent downward trend, with no upward trend at the low-expression end (left).
    • RNA-seq, when starting with 55k genes it is not uncommon to end up with ~15k genes or less post-filtering.
    • ATAC-seq consensus regions scale with the number of samples. Nevertheless, we had good experiences with ~100k genomic regions post-filtering.
• Normalize (norm{method}.csv)
  • The data can be normalized using several methods to correct for technical biases (e.g., differences in library size).
  • All methods supported in edgeR’s function CalcNormFactors with subequent CPM/RPKM quantification including method specific parameters can be configured.
  • CQN (Conditional Quantile Normalization) corrects for a covariate (e.g., GC-content) and feature-length biases (e.g., gene length). The QR fit of the covariate and feature-length are provided as plots (normCQN_QRfit.png).
  • VOOM (Mean-Variance Modeling at the Observational Level) from the package limma estimates the mean-variance relationship of the log counts and generates a precision weight for each observation. The Mean-Variance trend plot is provided (normVOOM_mean_variance_trend.png).
  • All normalization outputs are log2-normalized.
• Integrate (*_reComBat.csv)
  • The data can be integrated using the reComBat method, which requires log-normalized data.
  • This method adjusts for batch effects and unwanted sources of variation while trying to retain desired sources of variation e.g., biological variability.
  • This is particularly useful when combining data from different experiments or sequencing runs.
  • Use as few variables as possible for the (un)wanted parameters, as they often correlate (e.g., sequencing statistics) and can dilute the model’s predictive/corrective power across multiple variables.
  • For unwanted sources of variation, start with the strongest confounder; this is often sufficient.
  • For wanted sources of variation, combine all relevant metadata into a single column (e.g., condition) and use only this.
  • Note: Due to a reComBat bug, a numerical confounder can only be corrected if at least one categorical confounder is also declared.
    • Using the same variable for both batch and categorical confounder parameters can cause opposite batch effects.
    • We recommend addressing the numerical confounder in downstream analyses, such as within a differential analysis model.
• Highly Variable Feature Selection (*_HVF.csv)
  • The top percentage of the most variable features is selected based on the binned normalized dispersion of each feature adapted from Zheng (2017) Nature Communications.
  • These HVFs are often the most informative for downstream analyses such as clustering or differential expression, but smaller effects of interest could be lost.
  • The selection is visualized by histograms before and after normalization, mean to normalized dispersion scatterplots, and a scatterplot of the ranked normalized dispersion always highlighting the selected features (*_HVF_selection.png).
• Results ({split}/*.csv)
  • All transformed datasets are saved as CSV files and named by the applied methods, respectively.
  • Example: {split}/normCQN_reComBat_HVF.csv implies that the respective data {split} was filtered, normalized using CQN, integrated with reComBat and subset to its HVFs.
• Visualizations ({split}/plots/)
  • Next to the method-specific visualizations (e.g., for CQN, HVF selection), a diagnostic figure is provided for every generated dataset (*.png), consisting of the following plots:
    • Mean-Variance relationship of all features as a hexagonal heatmap of 2d bin counts.
    • Densities of log-normalized counts per sample colored by sample or configured annotation column.
    • Boxplots of log-normalized counts per sample colored by sample or configured annotation column.
    • Principal Component Analysis (PCA) plots, with samples colored by up to two annotation columns (e.g., batch and treatment).
  • Confounding Factor Analysis to inform integration (*_CFA.png)
    • Quantification of statistical association between provided metadata and (up to) the first ten principal components.
    • Categorical metadata association is tested using the non-parametric Kruskal-Wallis test, which is broadly applicable due to relaxed requirements and assumptions.
    • Numeric metadata association is tested using rank-based Kendall’s Tau, which is suitable for “small” data sets with many ties and is robust to outliers.
    • Statistical associations as -log10(adjusted p-values) are visualized using a heatmap with hierarchically clustered rows (metadata).
  • Correlation Heatmaps (*_heatmap_{clustered|sorted}.png)
    • Heatmap of sample-wise Pearson correlation matrix of the respective data split and processing step to quickly assess sample similarities e.g., replicates/conditions should correlate highly but batch shoud not.
    • Hierarchically clustered using method ‘complete’ with distance metric ‘euclidean’ (*_heatmap_clustered.png).
    • Alphabetically sorted by sample name (*_heatmap_sorted.png).
  • Note: raw and filtered counts are log2(x+1)-normalized for the visualizations.
  • These visualizations should help to assess the quality of the data and the effectiveness of the processing steps (e.g., normalization).
  • Visualizations are within each split’s plots subfolder, with the identical naming scheme as the respective data.

🛠️ Usage

Here are some tips for the usage of this workflow:

• Don’t be scared off by the number of configurable parameters, the goal was to enable maximum configurability, hence the config.yaml is quite comprehensive.
• Start with defaults, which are provided.
• Use a minimum of options and configuration changes at the beginning until the workflow is running, then start to adapt.
• Use the diagnostic visualizations to understand the effect different methods and parameter combinations have on your data.

⚙️ Configuration

Detailed specifications can be found here ./config/README.md

📖 Examples

— COMING SOON —

🔗 Links

• GitHub Repository
• GitHub Page
• Zenodo Repository
• Snakemake Workflow Catalog Entry

📚 Resources

• Recommended compatible MrBiomics modules
  • for upstream processing:
    • ATAC-seq Processing to quantify chromatin accessibility into count matrices as input.
    • scRNA-seq Data Processing & Visualization for processing (multimodal) single-cell transcriptome data. and creating pseudobulked count matrices as input.
  • for downstream analyses:
    • Unsupervised Analysis to understand and visualize similarities and variations between cells/samples, including dimensionality reduction and cluster analysis. Useful for all tabular data including single-cell and bulk sequencing data.
    • Differential Analysis with limma to identify and visualize statistically significantly different features (e.g., genes or genomic regions) between sample groups.
    • Enrichment Analysis for biomedical interpretation of (differential) analysis results using prior knowledge.
• Bioconductor - RNAseq123 - Workflow
• limma workflow tutorial RNA-seq analysis is easy as 1-2-3 with limma, Glimma and edgeR
  • notebook
  • paper
• Normalized dispersion calculation for selection of highly variable features adapted from Zheng (2017) Nature Communications.

📑 Publications

The following publications successfully used this module for their analyses.

• FirstAuthors et al. (202X) Journal Name - Paper Title.
• …

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