Analysis of Slide-seqV2 mouse olfactory bulb dataset

In this tutorial, we demonstrate SpaRCL on the analysis of Slide-seqV2 mouse olfactory bulb dataset including

• Spatial reconstruction

• Mini-batch relational contrastive learning

• Spatial domain identification

• Finding differentially expressed genes

• Spatial domain annotation

The dataset is available at Single Cell Portal (Puck_200127_15).

import numpy as np
import pandas as pd
import scanpy as sc
import matplotlib.pyplot as plt
import seaborn as sns

import SpaRCL as rcl

Data loading and preprocessing

We load the dataset and find top 8000 highly variable genes.

adata = sc.read_h5ad('./data/Puck_200127_15.h5ad')
adata

AnnData object with n_obs × n_vars = 21724 × 21220
    obsm: 'spatial'

sc.pp.highly_variable_genes(adata, n_top_genes=8000, flavor='seurat_v3')

Spatial reconstruction

We perform spatial reconstruction to aggregate expression from spatial neighbors.

rcl.spatial_reconstruction(adata, n_neighbors=30)

Mini-batch relational contrastive learning

We select 4000 reference centers and then perform mini-batch relational contrastive learning on the reconstructed data.

sc.pp.pca(adata)
rcl.reference_centers(adata, n_centers=4000)

C:\ProgramData\Anaconda3\lib\site-packages\sklearn\cluster\_kmeans.py:887: UserWarning: MiniBatchKMeans is known to have a memory leak on Windows with MKL, when there are less chunks than available threads. You can prevent it by setting batch_size >= 4096 or by setting the environment variable OMP_NUM_THREADS=1
  warnings.warn(

Since this step takes some time, we load previously computed AnnData.

# rcl.run_RCL_minibatch(adata)

adata = sc.read_h5ad('./data/Puck_200127_15_results.h5ad')
adata

AnnData object with n_obs × n_vars = 21724 × 21220
    obs: 'reference_centers'
    var: 'highly_variable', 'highly_variable_rank', 'means', 'variances', 'variances_norm'
    uns: 'hvg', 'relation', 'pca', 'reference_centers', 'spatial_reconstruction'
    obsm: 'X_pca', 'relation', 'spatial'
    varm: 'PCs'
    layers: 'counts', 'log1p'

Spatial domain identification

We create a temporary AnnData using the sample-by-reference relation matrix and then interoperate with SCANPY to identify spatial domains.

adata_V = sc.AnnData(adata.obsm['relation'])
adata_V.obs_names = adata.obs_names
adata_V.obsm['spatial'] = adata.obsm['spatial']
adata_V

AnnData object with n_obs × n_vars = 21724 × 3933
    obsm: 'spatial'

sc.pp.pca(adata_V)
sc.pp.neighbors(adata_V)
sc.tl.louvain(adata_V, resolution=0.4)

adata.obs['louvain'] = adata_V.obs['louvain']

We exclude domain 1 and 7 from downstream analysis, since their average counts are lower than 100.

sns.barplot(y=np.sum(adata.layers['counts'].toarray(), axis=1), x=adata.obs['louvain'])

<AxesSubplot:xlabel='louvain'>

adata_subset = adata[~adata.obs['louvain'].isin(['1', '7']),:]

fig, axs = plt.subplots(figsize=(8, 7))

sc.pl.embedding(
    adata_subset,
    basis='spatial',
    color='louvain',
    size=50,
    palette=sc.pl.palettes.default_102,
    legend_loc='right margin',
    show=False,
    ax=axs,
)

plt.tight_layout()

Trying to set attribute `.uns` of view, copying.

Finding differentially expressed genes

We find the differentially expressed (DE) genes across identified domains and show the domains and their DE gene expression patterns in spatial coordinates.

sc.tl.rank_genes_groups(adata_subset, groupby='louvain', use_raw=False, layer='counts', method='t-test_overestim_var')

de_genes = pd.DataFrame(adata_subset.uns['rank_genes_groups']['names']).iloc[:10,:]
de_genes

	0	2	3	4	5	6	8	9	10	11
0	Pcp4	mt-Nd4	Nrsn1	S100a5	Ptgds	Mbp	Cpne6	Gap43	Rab3b	S100a5
1	Camk2n1	Doc2g	Cck	Fabp7	Hbb-bs	Plp1	Hap1	Snca	Doc2g	Omp
2	Pcp4l1	mt-Nd1	S100a5	Apod	Hba-a1	Mobp	Macrod2	Doc2g	Map1b	Stoml3
3	Camk2b	mt-Cytb	mt-Cytb	Gng13	Hbb-bt	Cnp	Nos1	Uchl1	Slc17a7	Atf5
4	Meis2	mt-Rnr1	Calb2	Omp	Igf2	Mal	Synpr	Fxyd6	Atp1b1	Scgb1c1
5	Calm2	mt-Nd2	Olfm1	Atf5	Hba-a2	Cldn11	Tac1	Stmn2	Olfm1	Vmo1
6	Gng4	mt-Nd5	mt-Nd1	Npy	Mgp	Fth1	Pnmal2	Calb2	Syp	Cbr2
7	Malat1	mt-Co1	mt-Nd4	Ptn	Aebp1	Trf	Atp2b4	Rasgrp1	Stmn3	Tmsb4x
8	Nrxn3	mt-Rnr2	Trh	Kctd12	Col1a2	Mog	Nrxn3	Tuba1a	Thy1	Gng13
9	Ppp3ca	Slc6a11	Sparcl1	Stoml3	Slc6a20a	Mag	Necab2	Map1b	Shisa3	Calm1

fig, axs = plt.subplots(figsize=(8, 7))

sc.pl.embedding(
    adata_subset,
    basis='spatial',
    color='louvain',
    groups='0',
    layer='log1p',
    size=50,
    palette=sc.pl.palettes.default_102,
    legend_loc='right margin',
    show=False,
    ax=axs,
)

plt.tight_layout()

fig, axs = plt.subplots(figsize=(8, 7))

sc.pl.embedding(
    adata_subset,
    basis='spatial',
    color=de_genes.iloc[0,0],
    layer='log1p',
    size=50,
    color_map='Reds',
    vmax='p99.9',
    show=False,
    ax=axs,
)

plt.tight_layout()

fig, axs = plt.subplots(figsize=(8, 7))

sc.pl.embedding(
    adata_subset,
    basis='spatial',
    color='louvain',
    groups='4',
    layer='log1p',
    size=50,
    palette=sc.pl.palettes.default_102,
    legend_loc='right margin',
    show=False,
    ax=axs,
)

plt.tight_layout()

fig, axs = plt.subplots(figsize=(8, 7))

sc.pl.embedding(
    adata_subset,
    basis='spatial',
    color=de_genes.iloc[0,3],
    layer='log1p',
    size=50,
    color_map='Reds',
    vmax='p99.9',
    show=False,
    ax=axs,
)

plt.tight_layout()

Spatial domain annotation

We annotate the spatial domains based on the anatomical annotation from Allen Reference Atlas and marker genes.

map_dict = {
    '0': 'GCL',
    '2': 'EPL',
    '3': 'GL_1',
    '4': 'ONL',
    '5': 'GL_2',
    '6': 'RMS',
    '8': 'AOBgr',
    '9': 'AOB',
    '10': 'MCL',
    '11': 'UNK',
}

adata_subset.obs['annotation'] = adata_subset.obs['louvain'].map(map_dict).astype('category')

fig, axs = plt.subplots(figsize=(8.3, 7))

sc.pl.embedding(
    adata_subset,
    basis='spatial',
    color='annotation',
    size=50,
    palette=sc.pl.palettes.default_102,
    legend_loc='right margin',
    show=False,
    ax=axs,
)

plt.tight_layout()