Note

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Data loading and preparation

Here we walk through the necessary steps to get your data into ready for scvi-tools.

[2]:
import sys

#if branch is stable, will install via pypi, else will install from source
branch = "stable"
IN_COLAB = "google.colab" in sys.modules

if IN_COLAB and branch == "stable":
    !pip install --quiet scvi-tools[tutorials]
elif IN_COLAB and branch != "stable":
    !pip install --quiet --upgrade jsonschema
    !pip install --quiet git+https://github.com/yoseflab/scvi-tools@$branch#egg=scvi-tools[tutorials]
[3]:
import scvi
import scanpy as sc

Loading data

scvi-tools supports the AnnData data format, which also underlies Scanpy. AnnData is quite similar to other popular single cell objects like that of Seurat and SingleCellExperiment. In particular, it allows cell-level and feature-level metadata to coexist in the same data structure as the molecular counts.

It’s also now possible to automatically convert these R-based objects to AnnData within a Jupyter notebook. See the following tutorial for more information.

scvi-tools has a number of convenience methods for loading data from .csv, .loom, and .h5ad formats. To load ouputs from Cell Ranger, please use Scanpy’s reading functionality.

Let us now download an AnnData object (.h5ad format) and load it using scvi-tools.

PBMC3k

[3]:
!wget 'http://falexwolf.de/data/pbmc3k_raw.h5ad'
--2021-02-23 18:30:03--  http://falexwolf.de/data/pbmc3k_raw.h5ad
Resolving falexwolf.de (falexwolf.de)... 85.13.135.70
Connecting to falexwolf.de (falexwolf.de)|85.13.135.70|:80... connected.
HTTP request sent, awaiting response... 200 OK
Length: 5855727 (5.6M)
Saving to: 'pbmc3k_raw.h5ad'

pbmc3k_raw.h5ad     100%[===================>]   5.58M  2.85MB/s    in 2.0s

2021-02-23 18:30:05 (2.85 MB/s) - 'pbmc3k_raw.h5ad' saved [5855727/5855727]

[4]:
pbmc3k = scvi.data.read_h5ad("pbmc3k_raw.h5ad")
[5]:
pbmc3k
[5]:
AnnData object with n_obs × n_vars = 2700 × 32738
    var: 'gene_ids'

This is a fairly simple object, it just contains the count data and the ENSEMBL ids for the genes.

[6]:
pbmc3k.var.head()
[6]:
gene_ids
index
MIR1302-10 ENSG00000243485
FAM138A ENSG00000237613
OR4F5 ENSG00000186092
RP11-34P13.7 ENSG00000238009
RP11-34P13.8 ENSG00000239945

PBMC5k

As another example, let’s download a dataset from 10x Genomics. This data was obtained from a CITE-seq experiment, so it also contains protein count data.

[7]:
!wget https://cf.10xgenomics.com/samples/cell-exp/3.0.2/5k_pbmc_protein_v3/5k_pbmc_protein_v3_filtered_feature_bc_matrix.h5
--2021-02-23 18:30:07--  https://cf.10xgenomics.com/samples/cell-exp/3.0.2/5k_pbmc_protein_v3/5k_pbmc_protein_v3_filtered_feature_bc_matrix.h5
Resolving cf.10xgenomics.com (cf.10xgenomics.com)... 104.18.0.173, 104.18.1.173, 2606:4700::6812:ad, ...
Connecting to cf.10xgenomics.com (cf.10xgenomics.com)|104.18.0.173|:443... connected.
HTTP request sent, awaiting response... 200 OK
Length: 17129253 (16M) [binary/octet-stream]
Saving to: '5k_pbmc_protein_v3_filtered_feature_bc_matrix.h5'

5k_pbmc_protein_v3_ 100%[===================>]  16.33M  8.97MB/s    in 1.8s

2021-02-23 18:30:09 (8.97 MB/s) - '5k_pbmc_protein_v3_filtered_feature_bc_matrix.h5' saved [17129253/17129253]

[8]:
pbmc5k = sc.read_10x_h5(
    "5k_pbmc_protein_v3_filtered_feature_bc_matrix.h5",
    gex_only=False
)
Variable names are not unique. To make them unique, call `.var_names_make_unique`.

It’s often helpful to give the gene names unique names.

[9]:
pbmc5k.var_names_make_unique()

We can see that adata.X contains the concatenated gene and protein expression data.

[10]:
pbmc5k.var.feature_types.astype("category").cat.categories
[10]:
Index(['Antibody Capture', 'Gene Expression'], dtype='object')

We can use scvi-tools to organize this object, which places the protein expression in adata.obms["protein_expression].

[11]:
scvi.data.organize_cite_seq_10x(pbmc5k)
[12]:
pbmc5k
[12]:
AnnData object with n_obs × n_vars = 5247 × 33538
    var: 'gene_ids', 'feature_types', 'genome'
    obsm: 'protein_expression'

Concatenate the datasets

[13]:
adata = pbmc5k.concatenate(pbmc3k)

Notice that the resulting AnnData has a batch key in .obs.

[14]:
adata.obs.head()
[14]:
batch
AAACCCAAGAGACAAG-1-0 0
AAACCCAAGGCCTAGA-1-0 0
AAACCCAGTCGTGCCA-1-0 0
AAACCCATCGTGCATA-1-0 0
AAACGAAAGACAAGCC-1-0 0

Preprocessing the data

It is common to remove outliers, and even perform feature selection before model fitting. We prefer the Scanpy preprocessing module at this stage.

[15]:
sc.pp.filter_genes(adata, min_counts=3)
sc.pp.filter_cells(adata, min_counts=3)
/home/galen/.pyenv/versions/3.8.3/envs/scvi-dev/lib/python3.8/site-packages/pandas/core/arrays/categorical.py:2487: FutureWarning: The `inplace` parameter in pandas.Categorical.remove_unused_categories is deprecated and will be removed in a future version.
  res = method(*args, **kwargs)

As it is popular to use normalize the data for many methods, we can use Scanpy for this; however, it’s important to keep the count information intact for scvi-tools models.

[16]:
adata.layers["counts"] = adata.X.copy()

Now we can proceed with common normalization methods.

[17]:
sc.pp.normalize_total(adata, target_sum=1e4)
sc.pp.log1p(adata)

We can store the normalized values in .raw to keep them safe in the event the anndata gets subsetted feature-wise.

[18]:
adata.raw = adata

Register the data with scvi-tools

Now that we have an AnnData object, we need to alert scvi-tools of all the interesting data in our object. For example, now that we have batches in our AnnData, we can alert the models that we’d like to perform batch correction. Also, because we have the count data in a layer, we can use the layer argument.

Basic case

[19]:
scvi.data.setup_anndata(adata, layer="counts", batch_key="batch")
INFO     Using batches from adata.obs["batch"]
INFO     No label_key inputted, assuming all cells have same label
INFO     Using data from adata.layers["counts"]
INFO     Computing library size prior per batch
INFO     Successfully registered anndata object containing 7947 cells, 14309 vars, 2 batches,
         1 labels, and 0 proteins. Also registered 0 extra categorical covariates and 0 extra
         continuous covariates.
INFO     Please do not further modify adata until model is trained.
/home/galen/.pyenv/versions/3.8.3/envs/scvi-dev/lib/python3.8/site-packages/pandas/core/arrays/categorical.py:2487: FutureWarning: The `inplace` parameter in pandas.Categorical.remove_unused_categories is deprecated and will be removed in a future version.
  res = method(*args, **kwargs)
/home/galen/.pyenv/versions/3.8.3/envs/scvi-dev/lib/python3.8/site-packages/pandas/core/arrays/categorical.py:2487: FutureWarning: The `inplace` parameter in pandas.Categorical.remove_unused_categories is deprecated and will be removed in a future version.
  res = method(*args, **kwargs)

Notice the info messages notify us that batches were detected in the data. Just to demonstrate what happens if we don’t include this option:

[20]:
scvi.data.setup_anndata(adata, layer="counts")
INFO     No batch_key inputted, assuming all cells are same batch
INFO     No label_key inputted, assuming all cells have same label
INFO     Using data from adata.layers["counts"]
INFO     Computing library size prior per batch
INFO     Successfully registered anndata object containing 7947 cells, 14309 vars, 1 batches,
         1 labels, and 0 proteins. Also registered 0 extra categorical covariates and 0 extra
         continuous covariates.
INFO     Please do not further modify adata until model is trained.
/home/galen/.pyenv/versions/3.8.3/envs/scvi-dev/lib/python3.8/site-packages/pandas/core/arrays/categorical.py:2487: FutureWarning: The `inplace` parameter in pandas.Categorical.remove_unused_categories is deprecated and will be removed in a future version.
  res = method(*args, **kwargs)

Now integration-based tasks can no longer be performed in subsequent models because there is no knowledge of such information.

CITE-seq case

As PBMC5k is a CITE-seq dataset, we can use scvi-tools to register the protein expression. Note that totalVI is the only current model that uses the protein expression. The usage of registered items is model specific. As another example, registering the labels in the AnnData object will not affect totalVI or scVI, but is necessary to run scANVI.

We have not preprocessed the pbmc5k object, which we do recommend. We show how to run setup_anndata in this case for illustrative purposes.

[21]:
scvi.data.setup_anndata(pbmc5k, protein_expression_obsm_key="protein_expression")
INFO     No batch_key inputted, assuming all cells are same batch
INFO     No label_key inputted, assuming all cells have same label
INFO     Using data from adata.X
INFO     Computing library size prior per batch
INFO     Using protein expression from adata.obsm['protein_expression']
INFO     Using protein names from columns of adata.obsm['protein_expression']
INFO     Successfully registered anndata object containing 5247 cells, 33538 vars, 1 batches,
         1 labels, and 32 proteins. Also registered 0 extra categorical covariates and 0
         extra continuous covariates.
INFO     Please do not further modify adata until model is trained.

Warning

After setup_anndata has been run, the adata object should not be modified. In other words, the very next step in the workflow is to initialize and train the model of interest (e.g., scVI, totalVI). If you do modify the adata, it’s ok, just run setup_anndata again – and then reinitialize the model.

Viewing the scvi-tools data setup

[22]:
scvi.data.view_anndata_setup(pbmc5k)
Anndata setup with scvi-tools version 0.0.0.
              Data Summary              
┏━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━┳━━━━━━━┓
┃             Data              Count ┃
┡━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━╇━━━━━━━┩
│            Cells              5247  │
│             Vars              33538 │
│            Labels               1   │
│           Batches               1   │
│           Proteins             32   │
│ Extra Categorical Covariates    0   │
│ Extra Continuous Covariates     0   │
└──────────────────────────────┴───────┘
                   SCVI Data Registry                    
┏━━━━━━━━━━━━━━━━━━━━┳━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━┓
┃        Data               scvi-tools Location        ┃
┡━━━━━━━━━━━━━━━━━━━━╇━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━┩
│         X                       adata.X              │
│   batch_indices         adata.obs['_scvi_batch']     │
│    local_l_mean     adata.obs['_scvi_local_l_mean']  │
│    local_l_var       adata.obs['_scvi_local_l_var']  │
│       labels           adata.obs['_scvi_labels']     │
│ protein_expression  adata.obsm['protein_expression'] │
└────────────────────┴──────────────────────────────────┘
                        Label Categories                        
┏━━━━━━━━━━━━━━━━━━━━━━━━━━━┳━━━━━━━━━━━━┳━━━━━━━━━━━━━━━━━━━━━┓
┃      Source Location       Categories  scvi-tools Encoding ┃
┡━━━━━━━━━━━━━━━━━━━━━━━━━━━╇━━━━━━━━━━━━╇━━━━━━━━━━━━━━━━━━━━━┩
│ adata.obs['_scvi_labels']      0                0          │
└───────────────────────────┴────────────┴─────────────────────┘
                       Batch Categories                        
┏━━━━━━━━━━━━━━━━━━━━━━━━━━┳━━━━━━━━━━━━┳━━━━━━━━━━━━━━━━━━━━━┓
┃     Source Location       Categories  scvi-tools Encoding ┃
┡━━━━━━━━━━━━━━━━━━━━━━━━━━╇━━━━━━━━━━━━╇━━━━━━━━━━━━━━━━━━━━━┩
│ adata.obs['_scvi_batch']      0                0          │
└──────────────────────────┴────────────┴─────────────────────┘

Transferring the anndata setup

In this section we will transfer the setup from one AnnData object to another AnnData object.

[4]:
adata1 = scvi.data.synthetic_iid()
print(adata1)
INFO     Using batches from adata.obs["batch"]
INFO     Using labels from adata.obs["labels"]
INFO     Using data from adata.X
INFO     Computing library size prior per batch
INFO     Using protein expression from adata.obsm['protein_expression']
INFO     Using protein names from adata.uns['protein_names']
INFO     Successfully registered anndata object containing 400 cells, 100 vars, 2 batches, 3
         labels, and 100 proteins. Also registered 0 extra categorical covariates and 0 extra
         continuous covariates.
INFO     Please do not further modify adata until model is trained.
AnnData object with n_obs × n_vars = 400 × 100
    obs: 'batch', 'labels', '_scvi_batch', '_scvi_labels', '_scvi_local_l_mean', '_scvi_local_l_var'
    uns: 'protein_names', '_scvi'
    obsm: 'protein_expression'
/home/galen/.pyenv/versions/3.8.3/envs/scvi-dev/lib/python3.8/site-packages/pandas/core/arrays/categorical.py:2487: FutureWarning: The `inplace` parameter in pandas.Categorical.remove_unused_categories is deprecated and will be removed in a future version.
  res = method(*args, **kwargs)
/home/galen/.pyenv/versions/3.8.3/envs/scvi-dev/lib/python3.8/site-packages/pandas/core/arrays/categorical.py:2487: FutureWarning: The `inplace` parameter in pandas.Categorical.remove_unused_categories is deprecated and will be removed in a future version.
  res = method(*args, **kwargs)
[5]:
adata2 = scvi.data.synthetic_iid(run_setup_anndata=False)
print(adata2)
AnnData object with n_obs × n_vars = 400 × 100
    obs: 'batch', 'labels'
    uns: 'protein_names'
    obsm: 'protein_expression'

transfer_anndata_setup() will use the fields used to setup adata1 to register the data in adata2.

[6]:
scvi.data.transfer_anndata_setup(adata1,adata2)
INFO     Using data from adata.X
INFO     Computing library size prior per batch
INFO     Registered keys:['X', 'batch_indices', 'local_l_mean', 'local_l_var', 'labels',
         'protein_expression']
INFO     Successfully registered anndata object containing 400 cells, 100 vars, 2 batches, 3
         labels, and 100 proteins. Also registered 0 extra categorical covariates and 0 extra
         continuous covariates.
/home/galen/.pyenv/versions/3.8.3/envs/scvi-dev/lib/python3.8/site-packages/pandas/core/arrays/categorical.py:2487: FutureWarning: The `inplace` parameter in pandas.Categorical.remove_unused_categories is deprecated and will be removed in a future version.
  res = method(*args, **kwargs)
/home/galen/.pyenv/versions/3.8.3/envs/scvi-dev/lib/python3.8/site-packages/pandas/core/arrays/categorical.py:2487: FutureWarning: The `inplace` parameter in pandas.Categorical.remove_unused_categories is deprecated and will be removed in a future version.
  res = method(*args, **kwargs)
[ ]: