nadia-combine

Overview

Combine multiple single cell RNA-seq datasets.

From Orchestrating Single-Cell Analysis with Bioconductor book:: Large single-cell RNA sequencing (scRNA-seq) projects usually need to generate data across multiple batches/datasets due to logistical constraints. However, the processing of different batches is often subject to uncontrollable differences, e.g., changes in operator, differences in reagent quality. This results in systematic differences in the observed expression in cells from different batches, which we refer to as “batch effects”. Batch effects are problematic as they can be major drivers of heterogeneity in the data, masking the relevant biological differences and complicating interpretation of the results.

nadia-combine takes processed AnnData object (h5ad file), integrates and corrects for batch effects across multiple datasets. It provides two methods for data integration: harmony and scanorama.

Learn more about Data Integration, see this tutorial.

How it works

1. Detect variable genes

First, all anndata objects are concatenated into single object. The matrix in processed AnnData object stores logtransformed normalized counts, so we can use it directly to detect variable genes, and then perform PCA.

# compute variable genes
sc.pp.highly_variable_genes(adata, min_mean=0.0125, max_mean=3, min_disp=0.5)

# subset for variable genes in the dataset
adata = adata[:, adata.var['highly_variable']]

# scale data, clip values exceeding standard deviation 10.
sc.pp.scale(adata, max_value=10)

# PCA
sc.tl.pca(adata, svd_solver='arpack')

2. Data integration

Users can choose the integration method by -m, –method option.

# harmony
sce.pp.harmony_integrate(adata, 'sample')

# scanorama
sce.pp.scanorama_integrate(adata, 'sample')

After that, UMAP and T-SNE embeddings are built. We create UMAP and T-SNE plots with cell are color by sample, as well as violin plot of metadata by sample.

Input

Processed AnnData object (h5ad file) from nadia-process.

Output

Integrated AnnData object

Integrated AnnData object (allsample.h5ad), which could be used in cellxgene - visualization tool.

Report

nadia-combine produces a multiqc report in html format. You can download an example report

Plots

UMAP plot by sample

T-SNE plot by sample

Violin plots of metadata by sample

UMI count

../_images/violin_n_counts_by_sample.png

Gene count

Mitochondrial genes percentage

../_images/violin_Mito_percent_by_sample.png

Ribosomal genes percentage

../_images/violin_Ribo_percent_by_sample.png

Doublet score

Usage examples

nadia-combine \
    --h5ad processed_anndata/*_processed.h5ad \
    -o nadia-combine \
    -m harmony \
    --n-pcs 10

Argument details

Input/Output options

`--h5ad`

Required

Path to processed h5ad file (AnnData object)

`-o`, `--outdir`

Required

Output directory

Integrate options:

`-m, --method`

Choices: harmony, scanorama, None

Integration method. If None, then do not perform data intergration.

`--n-pcs`

Default: 30

Number of Principle Components to compute UMAP and tSNE.

`--n-neighbors`

Default: 20

Number of neighbors to compute UMAP.