Integration of CITE-seq and scRNA-seq data#

Here we demonstrate how to integrate CITE-seq and scRNA-seq datasets with totalVI. The same principles here can be used to integrate CITE-seq datasets with different sets of measured proteins.

Note

Running the following cell will install tutorial dependencies on Google Colab only. It will have no effect on environments other than Google Colab.

!pip install --quiet scvi-colab
from scvi_colab import install

install()
WARNING: Running pip as the 'root' user can result in broken permissions and conflicting behaviour with the system package manager, possibly rendering your system unusable.It is recommended to use a virtual environment instead: https://pip.pypa.io/warnings/venv. Use the --root-user-action option if you know what you are doing and want to suppress this warning.

import tempfile

import matplotlib.pyplot as plt
import numpy as np
import pandas as pd
import plotnine as p9
import scanpy as sc
import scvi
import seaborn as sns
import torch
from scipy.stats import pearsonr

Imports and data loading#

scvi.settings.seed = 0
print("Last run with scvi-tools version:", scvi.__version__)
Last run with scvi-tools version: 1.1.6

Note

You can modify save_dir below to change where the data files for this tutorial are saved.

sc.set_figure_params(figsize=(6, 6), frameon=False)
sns.set_theme()
torch.set_float32_matmul_precision("high")
save_dir = tempfile.TemporaryDirectory()

%config InlineBackend.print_figure_kwargs={"facecolor": "w"}
%config InlineBackend.figure_format="retina"

Here we focus on two CITE-seq datasets of peripheral blood mononuclear cells from 10x Genomics and used in the totalVI manuscript. We have already filtered these datasets for doublets and low-quality cells and genes.

The quality of totalVI’s protein imputation is somewhat reliant on how well the datasets mix in the latent space. In other words, it’s assumed here the datasets largely share the same cell subpopulations.

adata = scvi.data.pbmcs_10x_cite_seq(save_path=save_dir.name)
INFO     Downloading file at /tmp/tmpg1z68alw/pbmc_10k_protein_v3.h5ad
INFO     Downloading file at /tmp/tmpg1z68alw/pbmc_5k_protein_v3.h5ad
# batch 0 corresponds to dataset_10k, batch 1 corresponds to dataset_5k
batch = adata.obs.batch.values.ravel()
adata.obs.batch
index
AAACCCAAGATTGTGA-1    PBMC10k
AAACCCACATCGGTTA-1    PBMC10k
AAACCCAGTACCGCGT-1    PBMC10k
AAACCCAGTATCGAAA-1    PBMC10k
AAACCCAGTCGTCATA-1    PBMC10k
                       ...   
TTTGGTTGTACGAGTG-1     PBMC5k
TTTGTTGAGTTAACAG-1     PBMC5k
TTTGTTGCAGCACAAG-1     PBMC5k
TTTGTTGCAGTCTTCC-1     PBMC5k
TTTGTTGCATTGCCGG-1     PBMC5k
Name: batch, Length: 10849, dtype: object

Now we hold-out the proteins of the 5k dataset. To do so, we can replace all the values with 0s. We will store the original values to validate after training.

held_out_proteins = adata.obsm["protein_expression"][batch == "PBMC5k"].copy()
adata.obsm["protein_expression"].loc[batch == "PBMC5k"] = np.zeros_like(
    adata.obsm["protein_expression"][batch == "PBMC5k"]
)
sc.pp.highly_variable_genes(
    adata, batch_key="batch", flavor="seurat_v3", n_top_genes=4000, subset=True
)

Important

scvi-tools will automatically detect proteins as missing in a certain batch if the protein has 0 counts for each cell in the batch. In other words, to indicate a protein is missing in a certain batch, please set it to 0 for each cell.

scvi.model.TOTALVI.setup_anndata(
    adata, batch_key="batch", protein_expression_obsm_key="protein_expression"
)
INFO     Using column names from columns of adata.obsm['protein_expression']
INFO     Found batches with missing protein expression

Prepare and run model#

model = scvi.model.TOTALVI(adata, latent_distribution="normal", n_layers_decoder=2)
INFO     Computing empirical prior initialization for protein background.
model.train()
Monitored metric elbo_validation did not improve in the last 45 records. Best score: 1215.001. Signaling Trainer to stop.
plt.plot(model.history["elbo_train"], label="train")
plt.plot(model.history["elbo_validation"], label="val")
plt.title("Negative ELBO over training epochs")
plt.ylim(1100, 1500)
plt.legend()
<matplotlib.legend.Legend at 0x730f585e5d30>
../../../_images/40354c356d8d7b18e263a1d943d42559011b701889cc84e68367c8269e568f3d.png

Analyze outputs#

Again, we rely on Scanpy.

TOTALVI_LATENT_KEY = "X_totalVI"
PROTEIN_FG_KEY = "protein_fg_prob"

adata.obsm[TOTALVI_LATENT_KEY] = model.get_latent_representation()
adata.obsm[PROTEIN_FG_KEY] = model.get_protein_foreground_probability(transform_batch="PBMC10k")

rna, protein = model.get_normalized_expression(
    transform_batch="PBMC10k", n_samples=25, return_mean=True
)

Note

transform_batch is a powerful parameter. Setting this allows one to predict the expression of cells as if they came from the inputted batch. In this case, we’ve observed protein expression in batch “PBMC10k” (batch categories from original adata object), but we have no protein expression in batch “PBMC5k”. We’d like to take the cells of batch “PBMC5k” and make a counterfactual prediction: “What would the expression look like if my batch “PBMC5k” cells came from batch “PBMC10k”?”

protein.iloc[:5, :5]
CD3_TotalSeqB CD4_TotalSeqB CD8a_TotalSeqB CD14_TotalSeqB CD15_TotalSeqB
index
AAACCCAAGATTGTGA-1 14.775918 231.680618 1.632642 907.201538 3.478770
AAACCCACATCGGTTA-1 31.135048 180.610703 3.638931 733.734985 2.319544
AAACCCAGTACCGCGT-1 11.875708 400.952393 3.752194 1282.633789 15.500657
AAACCCAGTATCGAAA-1 1.739256 0.842190 23.565744 0.089554 71.286102
AAACCCAGTCGTCATA-1 0.466409 0.054686 60.923546 0.033670 0.221310

Important

The following is for illustrative purposes. In the code blocks above, we have the denoised protein values for each cell. These values have the expected protein background component removed. However, to compare to the held out protein values, we must include both protein foreground and background. We recommend using the values above for downstream tasks.

_, protein_means = model.get_normalized_expression(
    n_samples=25,
    transform_batch="PBMC10k",
    include_protein_background=True,
    sample_protein_mixing=False,
    return_mean=True,
)
TOTALVI_CLUSTERS_KEY = "leiden_totalVI"

sc.pp.neighbors(adata, use_rep=TOTALVI_LATENT_KEY)
sc.tl.umap(adata, min_dist=0.4)
sc.tl.leiden(adata, key_added=TOTALVI_CLUSTERS_KEY)
perm_inds = np.random.permutation(len(adata))
sc.pl.umap(
    adata[perm_inds],
    color=[TOTALVI_CLUSTERS_KEY, "batch"],
    ncols=1,
    frameon=False,
)
batch = adata.obs.batch.values.ravel()
combined_protein = np.concatenate(
    [adata.obsm["protein_expression"].values[batch == "PBMC10k"], held_out_proteins],
    axis=0,
)

# cleaner protein names
parsed_protein_names = [p.split("_")[0] for p in adata.obsm["protein_expression"].columns]
for i, p in enumerate(parsed_protein_names):
    adata.obs[f"{p} imputed"] = protein_means.iloc[:, i]
    adata.obs[f"{p} observed"] = combined_protein[:, i]
viz_keys = []
for p in parsed_protein_names:
    viz_keys.append(p + " imputed")
    viz_keys.append(p + " observed")

sc.pl.umap(
    adata[adata.obs.batch == "PBMC5k"],
    color=viz_keys,
    ncols=2,
    vmax="p99",
    frameon=False,
    add_outline=True,
    wspace=0.1,
)

Imputed vs denoised correlations#

imputed_pros = protein_means[batch == "PBMC5k"]
held_vs_denoised = pd.DataFrame()
held_vs_denoised["Observed (log)"] = np.log1p(held_out_proteins.values.ravel())
held_vs_denoised["Imputed (log)"] = np.log1p(imputed_pros.to_numpy().ravel())
protein_names_corrs = []
for i in range(len(parsed_protein_names)):
    protein_names_corrs.append(
        parsed_protein_names[i]
        + ": Corr="
        + str(np.round(pearsonr(held_out_proteins.values[:, i], imputed_pros.iloc[:, i])[0], 3))
    )
held_vs_denoised["Protein"] = protein_names_corrs * len(held_out_proteins)
held_vs_denoised.head()
Observed (log) Imputed (log) Protein
0 3.258097 3.596324 CD3: Corr=0.778
1 5.105945 5.966447 CD4: Corr=0.875
2 2.833213 3.539591 CD8a: Corr=0.821
3 6.546785 7.227937 CD14: Corr=0.913
4 2.995732 4.812809 CD15: Corr=0.058

We notice that CD15 has a really low correlation (imputation accuracy). Recall that imputation involves a counterfactual query – “what would the protein expression have been for these cells if they came from the PBMC10k dataset?” Thus, any technical issues with proteins in CD15 in PBMC10k will be reflected in the imputed values. It’s the case here that CD15 was not captured as well in the PBMC10k dataset compared to the PBMC5k dataset.

p9.theme_set(p9.theme_classic)
(
    p9.ggplot(held_vs_denoised, p9.aes("Observed (log)", "Imputed (log)"))
    + p9.geom_point(size=0.5)
    + p9.facet_wrap("~Protein", scales="free")
    + p9.theme(
        figure_size=(10, 10),
        panel_spacing=0.05,
    )
)