April 26 revisions

JackNg88 · Apr 26, 2020 · 59adeff · 59adeff
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diff --git a/airway_smoking_2020_gse134174.py b/airway_smoking_2020_gse134174.py
@@ -0,0 +1,209 @@
+#!/usr/bin/env python3
+# -*- coding: utf-8 -*-
+"""
+Created on Tue Apr 14 13:38:41 2020
+
+@author: Joan Smith
+https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE134174
+"""
+#%%
+
+import pandas as pd
+import scanpy as sc
+from matplotlib import pyplot as plt 
+import matplotlib as mpl
+import os
+import numpy as np
+#%%
+sc.settings.figdir = 'data/cluster-plots/airway-smoking-2020-gse134174/'
+sc.settings.verbosity = 4
+
+mpl.rcParams.update(mpl.rcParamsDefault)
+mpl.rcParams.update({
+    'font.sans-serif': 'Arial',
+    'font.family': 'sans-serif',
+    'axes.titlesize': 18,
+    'axes.labelsize': 14,
+    })
+
+#%% Load prenormed data and metadata
+input_file = 'data/raw-data/airway-smoking-GSE134174/GSE134174_Processed_invivo_norm.txt'
+metadata = pd.read_csv('data/raw-data/airway-smoking-GSE134174/GSE134174_Processed_invivo_metadata.txt', sep='\t', index_col=0)
+adata = sc.read_text(input_file).T
+adata.obs = metadata
+all_adata = adata.copy()
+VARIANT = 'all'
+
+#%%
+def exploratory_plots(adata):
+
+    num_non_int = (adata.to_df().applymap(float.is_integer) == False).sum().sum()
+    print('Num non-int: ', num_non_int)
+    plt.figure()
+    sc.pp.filter_cells(adata, min_genes=0)
+    plt.hist(adata.obs.n_genes, bins=500)
+    plt.title('Genes per cell')    
+    minimum = adata.obs['n_genes'].min()
+    plt.xlabel('# Genes. Min:' + str(minimum))
+    plt.ylabel('# Cells')
+
+    plt.figure()
+    sc.pl.highest_expr_genes(adata, n_top=20, )
+    sc.pl.pca_variance_ratio(adata, log=True)
+    plt.show()    
+exploratory_plots(adata)
+
+#%% HEAVY AND NEVER
+adata = all_adata[all_adata.obs.Smoke_status.isin(['heavy', 'never'])].copy()
+VARIANT='heavy_and_never'
+sc.settings.figdir = 'data/cluster-plots/airway-smoking-2020-gse134174-heavy-and-never'
+
+#%%
+sc.pp.highly_variable_genes(adata)
+sc.tl.pca(adata)
+sc.pp.neighbors(adata)
+
+#%%
+LEARNING_RATE = 1000
+EARLY_EXAGGERATION = 12
+RESOLUTION = 2.5 #2 
+PERPLEXITY=30
+N_PCS = 15
+
+sc.tl.tsne(adata, learning_rate=LEARNING_RATE, n_jobs=8, early_exaggeration=EARLY_EXAGGERATION, perplexity=PERPLEXITY, n_pcs=N_PCS)
+sc.tl.leiden(adata, resolution=RESOLUTION)
+
+params = {'learning_rate': LEARNING_RATE,
+          'early_exaggeration':EARLY_EXAGGERATION,
+          'resolution': RESOLUTION,
+          'perplexity': PERPLEXITY,
+          'n_pcs': N_PCS,
+          'genes': 'all',
+          'variant': VARIANT,
+          'files': input_file}
+sc.pl.tsne(adata, color=['leiden'], size=25, save='_leiden.pdf', color_map='plasma')
+pd.Series(params).to_csv(os.path.join(sc.settings.figdir, 'params.txt'))
+adata.uns['joan_cluster_params'] = params
+adata.write(os.path.join(sc.settings.figdir, 'adata.h5ad'))
+#%%
+#%%
+#%%
+#%%
+markers = pd.read_csv(os.path.join(sc.settings.figdir, 'markers.csv'), header=None, names=['gene', 'cluster'])
+markers['gene'] = markers['gene'].str.upper()
+markers = markers[markers['gene'].isin(adata.var.index.values)]
+
+markers['title'] = markers['gene'] + '+: ' + markers['cluster']
+markers = markers.set_index('gene')
+markers.loc['PTPRC']['title'] = 'PTPRC (CD45)+: Immune Cells'
+markers.loc['leiden'] = ['Leiden', 'Clusters']
+#%%
+adata = sc.read_h5ad(os.path.join(sc.settings.figdir, 'adata.h5ad'))
+params = pd.read_csv(os.path.join(sc.settings.figdir, 'params.txt'), index_col=0).to_dict('dict')['0']
+VARIANT = params['variant']
+#%%
+for i, g in markers.iterrows():
+    sc.pl.tsne(adata, color=i,
+           title=g['title'],
+           color_map='plasma',
+           size=25,
+           save='_' + i + '_airway_smoking_2020.pdf',
+           show=False)
+
+#%%
+sc.tl.rank_genes_groups(adata, 'leiden', method='t-test', n_genes=20)
+pd.DataFrame(adata.uns['rank_genes_groups']['names']).to_csv(os.path.join(sc.settings.figdir, 'rank_groups.csv'))
+adata.write(os.path.join(sc.settings.figdir, 'adata.h5ad'))
+
+
+#%%
+genes = ['ACE2', 'KRT5', 'TP63', 'BCAM', 'NGFR']
+df = adata.to_df()
+for g in genes:
+    print(g)
+    g_df = df.join(adata.obs['Smoke_status'])[[g, 'Smoke_status']]
+    g_df = g_df[g_df[g] > 0].pivot(columns='Smoke_status')
+    g_df.to_csv(os.path.join(sc.settings.figdir, g +'_smoking_status.csv'))
+
+#%%
+
+#%% Correlations
+
+df = adata.to_df()
+corr = df.corrwith(df['ACE2']).sort_values(ascending=False)
+print('correlations with ACE2')
+print(corr.head(20)) 
+corr.to_csv(os.path.join(sc.settings.figdir, 'ACE2_correlates.csv'))   
+
+
+#%%
+markers_for_pos_counts = markers.drop('leiden').copy()
+markers_for_pos_counts.loc['MKI67'] = ['MKI67', 'MKI67']
+markers_for_pos_counts.loc['PCNA'] = ['PCNA', 'PCNA']
+markers_for_pos_counts.loc['TOP2A'] = ['TOP2A', 'TOP2A']
+
+## pos for every marker, # cells
+df = adata.to_df()
+pos_counts = (df[markers_for_pos_counts.index] > 0).sum()
+pos_counts['cell_counts'] = len(df.index)
+pos_counts.to_csv(os.path.join(sc.settings.figdir, 'single_positive_counts.csv'), header=['single pos counts'])
+
+# # double pos w ace2, # 
+double_pos =  (df[markers_for_pos_counts.index] > 0).apply(lambda x: x & (df['ACE2'] > 0)).sum()
+double_pos['cell_counts'] = len(df.index)
+double_pos.to_csv(os.path.join(sc.settings.figdir, 'double_positive_counts.csv'), header=['double pos counts'])
+
+#%%
+df = adata.to_df()
+ace2_muc5ac_double_pos = (df['MUC5AC'] > 0) & (df['ACE2'] > 0)
+ace2_muc5ac = df.loc[ace2_muc5ac_double_pos][['ACE2']].join(adata.obs.Smoke_status)
+ace2_muc5ac.pivot(columns='Smoke_status').to_csv(os.path.join(sc.settings.figdir, 'ACE2_MUC5AC_double_pos.csv'))
+
+#%%
+cluster_labels = pd.read_csv(os.path.join(sc.settings.figdir, 'key.csv'), header=None, names=['cluster', 'label'], dtype='str')
+cluster_labels.loc[cluster_labels['label'].isna(),'label'] = cluster_labels.loc[cluster_labels['label'].isna(), 'cluster']
+cluster_labels_dict = cluster_labels.set_index('cluster')['label'].to_dict()
+adata.obs['Cell Types'] = adata.obs.leiden.map(cluster_labels_dict).astype('category')
+
+sc.pl.tsne(adata, color=['Cell Types'], size=25, save='_manually_labeled.pdf',  color_map='plasma', title='Human Trachea')
+adata.write(os.path.join(sc.settings.figdir, 'adata.h5ad'))
+
+sc.tl.rank_genes_groups(adata, 'Cell Types', method='t-test', n_genes=100, key_added='cell_types_gene_groups')
+pd.DataFrame(adata.uns['cell_types_gene_groups']['names']).to_csv(os.path.join(sc.settings.figdir, 'manually_labeled_rank_groups_ttest.csv'))
+
+#%%
+adata.obs.groupby('Cell Types')['Smoke_status'].value_counts().to_csv(os.path.join(sc.settings.figdir, 'cell_type_smoker_counts.csv'))
+ace2_pos_cell_types = adata.to_df()[(adata.to_df()['ACE2'] > 0)][['ACE2']].join(adata.obs[['Cell Types', 'Smoke_status']])
+ace2_pos_cell_types.groupby(['Cell Types', 'Smoke_status']).count().to_csv(os.path.join(sc.settings.figdir, 'cell_type_ace2_pos_counts.csv'))
+
+#%%
+adata.obs.loc[(adata.to_df()['ACE2'] > 0), 'ace2_pos'] = 'ace2_pos'
+adata.obs.loc[(adata.to_df()['ACE2'] <= 0), 'ace2_pos'] = 'ace2_neg'
+sc.tl.rank_genes_groups(adata, groupby='ace2_pos', key_added='ACE2_pos')
+#%%
+
+adata.obs['ACE2_goblet'] = np.nan
+adata.obs.loc[(adata.obs['Cell Types'] == 'Goblet/Club cells'), 'ACE2_goblet'] = 'Goblet/Club'
+adata.obs.loc[(adata.obs['Cell Types'] == 'Ciliated cells'), 'ACE2_goblet'] = 'Ciliated'
+adata.obs.loc[(adata.obs['Cell Types'] == 'Basal cells'), 'ACE2_goblet'] = 'Basal'
+adata.obs.loc[(adata.to_df()['ACE2'] > 0), 'ACE2_goblet'] = 'ACE2+'
+
+
+adata_ace2_goblet = adata[adata.obs.dropna(subset=['ACE2_goblet']).index].copy()
+adata_ace2_goblet.obs['ACE2_goblet'] = adata_ace2_goblet.obs['ACE2_goblet'].astype(pd.CategoricalDtype(ordered=True))
+adata_ace2_goblet.obs['ACE2_goblet'] = adata_ace2_goblet.obs['ACE2_goblet'].cat.reorder_categories(['ACE2+', 'Goblet/Club', 'Ciliated', 'Basal'], ordered=True)
+
+
+heatmap_genes = {'ACE2+ markers': adata.uns['ACE2_pos']['names']['ace2_pos'][1:11],
+                 'Goblet/Club markers': adata.uns['cell_types_gene_groups']['names']['Goblet/Club cells'][0:10],
+                 'Ciliated markers': adata.uns['cell_types_gene_groups']['names']['Ciliated cells'][0:10],
+                 'Basal markers': adata.uns['cell_types_gene_groups']['names']['Basal cells'][0:10],
+                 }
+
+ax = sc.pl.dotplot(adata_ace2_goblet, groupby='ACE2_goblet', var_names=heatmap_genes, #save='_ACE2_marker_goblet_ciliated.png', 
+              mean_only_expressed=True, var_group_rotation=0.0, show=False, figsize=(12, 2.5))
+all_axes = plt.gcf().get_axes()
+all_axes[0].set_ylabel('')
+
+plt.savefig(os.path.join(sc.settings.figdir, 'dotplot_ace2_marker_goblet_ciliated_basal.pdf'), bbox_inches='tight')
+
diff --git a/data/cluster-plots/airway-smoking-20200-gse134174/key.csv b/data/cluster-plots/airway-smoking-20200-gse134174/key.csv
@@ -0,0 +1,27 @@
+0,
+1,
+2,Basal cells
+3,Proliferating cells
+4,Goblet/Club cells
+5,Proliferating cells
+6,SMG-basal
+7,SMG-basal
+8,KRT8+ differentiating cells
+9,
+10,
+11,Ciliated cells
+12,Goblet/Club cells
+13,Basal cells
+14,
+15,Ciliated cells
+16,KRT8+ differentiating cells
+17,Basal cells
+18,Goblet/Club cells
+19,KRT8+ differentiating cells
+20,Goblet/Club cells
+21,Goblet/Club cells
+22,SMG-secretory
+23,Basal cells
+24,
+25,Ionocytes/Neuroendocrine cells
+26,Ionocytes/Neuroendocrine cells
diff --git a/data/cluster-plots/airway-smoking-20200-gse134174/markers.csv b/data/cluster-plots/airway-smoking-20200-gse134174/markers.csv
@@ -0,0 +1,80 @@
+ACE2,Coronavirus Receptor
+EPCAM,Epithelium
+PTPRC,Immune Cells
+PDGFRA,Mesenchymal Cells
+TMEM100,Endothelium
+RTKN2,Alveolar Type 1
+FOXJ1,Ciliated Cells
+LAMP3,Alveolar Type 2
+MUC5B,Secretory Cells
+MUC5AC,Goblet Cells
+SCGB3C1,Secretory Cells
+SCGB1A1,Club Cells
+GABRP,Secretory Cells
+LYPD2,Goblet Cells
+CLDN10,Secretory Cells
+KRT5,Basal Cells
+TRP63,Basal Cells
+MKI67,Proliferating Cells
+TOP2A,Proliferating Cells
+PCNA,Proliferating Cells
+DEUP1,Deuterosomal Cells
+FOXI1,Ionocytes
+CFTR,Ionocytes
+CGRP,Neuroendocrine Cells
+CHGA,Neuroendocrine Cells
+ASCL1,Brush/Tuft Cells
+LRMP,Brush/Tuft Cells
+COL1A1,Fibroblasts
+FBLN1,Fibroblasts
+ACTA2,Smooth Muscle Cells
+MYH11,Smooth Muscle Cells
+DNAH11,Ciliated Cells
+TP63,Basal Cells
+KRT8,Basal Cells
+CAV1,Basal Cells
+AGR2,Goblet Cells
+BPIFB1,Goblet/Club Cells
+SPDEF,Secretory Cells
+MUC1,Secretory Cells
+MUCL1,Secretory Cells?
+KIAA0101,01_p.basal
+TYMS,01_p.basal
+TK1,01_p.basal
+ZWINT,01_p.basal
+RRM2,01_p.basal
+KRT17,02_d.basal
+IL33,02_d.basal
+MMP10,02_d.basal
+BCAM,02_d.basal
+ETS2,02_d.basal
+C19orf33,03_KRT8.high
+GPRC5A,03_KRT8.high
+BHLHE40,03_KRT8.high
+TXN,03_KRT8.high
+EPAS1,03_KRT8.high
+BPIFB1,04_muc.secretory
+VMO1,04_muc.secretory
+MSMB,04_muc.secretory
+S100P,04_muc.secretory
+RDH10,04_muc.secretory
+FAM216B,05_ciliated
+STOML3,05_ciliated
+MRLN,05_ciliated
+CATSPERD,05_ciliated
+ANKRD66,05_ciliated
+HEPACAM2,06_rare
+PROX1,06_rare
+TUBB2B,06_rare
+NEURL1,06_rare
+PHGR1,06_rare
+KRT14,07_SMG.basal
+CAV1,07_SMG.basal
+FOS,07_SMG.basal
+G0S2,07_SMG.basal
+NAP1L1,07_SMG.basal
+SLPI,08_SMG.secretory
+RARRES1,08_SMG.secretory
+SCGB3A1,08_SMG.secretory
+TCN1,08_SMG.secretory
+MMP7,08_SMG.secretory
diff --git a/data/cluster-plots/gse1229560_ipf/key.csv b/data/cluster-plots/gse1229560_ipf/key.csv
@@ -0,0 +1,43 @@
+0,T cells
+1,Alveolar type 2
+2,Macrophages
+3,Macrophages
+4,Plasma cells
+5,Macrophages
+6,Alveolar type 2
+7,Macrophages
+8,Macrophages
+9,Macrophages
+10,Macrophages
+11,Ciliated cells
+12,Dendritic cells
+13,Macrophages
+14,Goblet/Club cells
+15,Endothelial cells
+16,B cells
+17,Proliferating cells
+18,B cells
+19,Alveolar type 2
+20,Macrophages
+21,Basal cells
+22,B cells
+23,Goblet/Club cells
+24,Macrophages
+25,Mast cells
+26,Macrophages
+27,Macrophages
+28,Macrophages
+29,Macrophages
+30,Macrophages
+31,Mesenchymal cells
+32,Alveolar type 2
+33,Smooth muscle cells
+34,T cells
+35,Endothelial cells
+36,Macrophages
+37,Macrophages
+38,Plasma cells
+39,Endothelial cells
+40,Macrophages
+41,Macrophages
+42,Mesenchymal cells
diff --git a/data/cluster-plots/healthy-mouse/key.csv b/data/cluster-plots/healthy-mouse/key.csv
@@ -0,0 +1,30 @@
+0,B cells
+1,Alveolar type 2
+2,T cells
+3,Macrophages
+4,NK cells
+5,Endothelial cells
+6,T cells
+7,Monocytes
+8,Monocytes
+9,Dendritic cells
+10,Endothelial cells
+11,Fibroblasts
+12,Macrophages
+13,Alveolar type 1
+14,Goblet/Club cells
+15,Endothelial cells
+16,B cells
+17,Alveolar type 2
+18,Neutrophils
+19,Ciliated cells
+20,Macrophages
+21,Endothelial cells
+22,Alveolar type 2
+23,Dendritic cells
+24,Alveolar type 2
+25,Monocytes
+26,Monocytes
+27,Alveolar type 2
+28,Neutrophils
+29,Alveolar type 2