Pristina atlas analysis 3 preprocessing.ipynb

{"cells":[{"cell_type":"markdown","id":"72ece39b","metadata":{"id":"72ece39b"},"source":["# Importing modules and settings"]},{"cell_type":"code","execution_count":null,"id":"238c8e41","metadata":{"id":"238c8e41"},"outputs":[],"source":["import numpy as np\n","import pandas as pd\n","import scanpy as sc"]},{"cell_type":"code","execution_count":null,"id":"a6ca374b","metadata":{"id":"a6ca374b"},"outputs":[],"source":["from matplotlib.pyplot import rc_context"]},{"cell_type":"code","execution_count":null,"id":"qJmDcET9-aIl","metadata":{"id":"qJmDcET9-aIl"},"outputs":[],"source":["import seaborn as sns"]},{"cell_type":"markdown","id":"4133c199","metadata":{"id":"4133c199"},"source":["General settings of Scanpy"]},{"cell_type":"code","execution_count":null,"id":"3be21d53","metadata":{"id":"3be21d53"},"outputs":[],"source":["sc.settings.verbosity = 4\n","sc.logging.print_header()\n","sc.settings.set_figure_params(dpi=80, facecolor='white')\n"]},{"cell_type":"code","execution_count":null,"id":"uEx2eXdl-ZG4","metadata":{"id":"uEx2eXdl-ZG4"},"outputs":[],"source":["umap_cmap = sns.light_palette('xkcd:medium blue', as_cmap = True)"]},{"cell_type":"markdown","id":"37ab1998","metadata":{"id":"37ab1998"},"source":["# Declaring the input and output files"]},{"cell_type":"code","execution_count":null,"id":"292c5538","metadata":{"id":"292c5538"},"outputs":[],"source":["name_of_analysis = 'pristina_atlas'"]},{"cell_type":"code","execution_count":null,"id":"affc908f","metadata":{"id":"affc908f"},"outputs":[],"source":["sc.settings.figdir = './figures/'+name_of_analysis+'_preprocessing'"]},{"cell_type":"code","execution_count":null,"id":"39f3c747","metadata":{"id":"39f3c747"},"outputs":[],"source":["results_file = './'+name_of_analysis+'.h5ad'"]},{"cell_type":"code","execution_count":null,"id":"4fe76765","metadata":{"id":"4fe76765"},"outputs":[],"source":["adata = sc.read_h5ad('./'+name_of_analysis+'_prefiltering.h5ad')"]},{"cell_type":"code","execution_count":null,"id":"8cb2aabc","metadata":{"id":"8cb2aabc"},"outputs":[],"source":["adata"]},{"cell_type":"code","execution_count":null,"id":"0jH8uMSF5mWg","metadata":{"id":"0jH8uMSF5mWg"},"outputs":[],"source":["adata.layers['counts'] = adata.X.copy()"]},{"cell_type":"code","execution_count":null,"id":"uTtqy6W85mZR","metadata":{"id":"uTtqy6W85mZR"},"outputs":[],"source":[]},{"cell_type":"markdown","id":"eca28d9f","metadata":{"id":"eca28d9f"},"source":["# Preprocessing"]},{"cell_type":"code","execution_count":null,"id":"bbccc3bd","metadata":{"id":"bbccc3bd"},"outputs":[],"source":["sc.pl.highest_expr_genes(adata, n_top=20, save = True)"]},{"cell_type":"code","execution_count":null,"id":"5bb0deb7","metadata":{"id":"5bb0deb7"},"outputs":[],"source":["sc.pp.filter_cells(adata, min_counts=50)\n","sc.pp.filter_cells(adata, min_genes= 50)\n","sc.pp.filter_genes(adata, max_counts = 1000000)"]},{"cell_type":"code","execution_count":null,"id":"d236f397","metadata":{"id":"d236f397"},"outputs":[],"source":["adata.var"]},{"cell_type":"code","execution_count":null,"id":"9a5dbb67","metadata":{"id":"9a5dbb67"},"outputs":[],"source":["sc.pl.highest_expr_genes(adata, n_top=20, save = True)"]},{"cell_type":"code","execution_count":null,"id":"458f1bcd","metadata":{"id":"458f1bcd"},"outputs":[],"source":["adata.var['mt'] = adata.var_names.str.startswith('MT-')  # annotate the group of mitochondrial genes as 'mt'\n","sc.pp.calculate_qc_metrics(adata, qc_vars=['mt'], percent_top=None, log1p=False, inplace=True)"]},{"cell_type":"code","execution_count":null,"id":"684ca625","metadata":{"id":"684ca625"},"outputs":[],"source":["sc.pl.violin(adata, ['n_genes_by_counts', 'total_counts', 'pct_counts_mt'],\n","             jitter= 0.7, multi_panel=True, log = True, save = True)"]},{"cell_type":"code","execution_count":null,"id":"ecd92371","metadata":{"id":"ecd92371"},"outputs":[],"source":["adata"]},{"cell_type":"markdown","id":"90f07840","metadata":{"id":"90f07840"},"source":["# Matrix slicing"]},{"cell_type":"code","execution_count":null,"id":"b86c5fea","metadata":{"id":"b86c5fea","scrolled":true},"outputs":[],"source":["sc.pl.scatter(adata, x='total_counts', y='n_genes_by_counts')"]},{"cell_type":"code","execution_count":null,"id":"74686eaa","metadata":{"id":"74686eaa"},"outputs":[],"source":["adata = adata[adata.obs.n_genes_by_counts < 700, :]"]},{"cell_type":"code","execution_count":null,"id":"dc9ee576","metadata":{"id":"dc9ee576"},"outputs":[],"source":["adata = adata[adata.obs.total_counts < 900, :]"]},{"cell_type":"code","execution_count":null,"id":"d61c9e7a","metadata":{"id":"d61c9e7a"},"outputs":[],"source":["sc.pl.scatter(adata, x='total_counts', y='n_genes_by_counts', save = True)"]},{"cell_type":"code","execution_count":null,"id":"081e4cb5","metadata":{"id":"081e4cb5"},"outputs":[],"source":[]},{"cell_type":"code","execution_count":null,"id":"7ef1e868","metadata":{"id":"7ef1e868"},"outputs":[],"source":[]},{"cell_type":"markdown","id":"cba76180","metadata":{"id":"cba76180"},"source":["# Normalization and log transformation"]},{"cell_type":"markdown","id":"dcc63620","metadata":{"id":"dcc63620"},"source":["The following 2 functions normalise and log transform the matrix"]},{"cell_type":"code","execution_count":null,"id":"afb05003","metadata":{"id":"afb05003"},"outputs":[],"source":["sc.pp.normalize_total(adata, target_sum=1e4)"]},{"cell_type":"code","execution_count":null,"id":"1888c724","metadata":{"id":"1888c724"},"outputs":[],"source":["sc.pp.log1p(adata)"]},{"cell_type":"markdown","id":"679d9115","metadata":{"id":"679d9115"},"source":["# Selecting highly variable genes"]},{"cell_type":"code","execution_count":null,"id":"88018c6c","metadata":{"id":"88018c6c"},"outputs":[],"source":["sc.pp.highly_variable_genes(adata, n_top_genes = 18000)"]},{"cell_type":"code","execution_count":null,"id":"4ae4f3db","metadata":{"id":"4ae4f3db"},"outputs":[],"source":["sc.pl.highly_variable_genes(adata, save = True)"]},{"cell_type":"code","execution_count":null,"id":"0cf960ce","metadata":{"id":"0cf960ce"},"outputs":[],"source":["adata.raw = adata"]},{"cell_type":"code","execution_count":null,"id":"0410daff","metadata":{"id":"0410daff"},"outputs":[],"source":["adata = adata[:, adata.var.highly_variable]"]},{"cell_type":"code","execution_count":null,"id":"21463047","metadata":{"id":"21463047"},"outputs":[],"source":["adata"]},{"cell_type":"code","execution_count":null,"id":"d3a3ba75","metadata":{"id":"d3a3ba75"},"outputs":[],"source":["adata.raw.var"]},{"cell_type":"code","execution_count":null,"id":"b613499b","metadata":{"id":"b613499b"},"outputs":[],"source":[]},{"cell_type":"markdown","id":"e4697b64","metadata":{"id":"e4697b64"},"source":["# Scaling the data"]},{"cell_type":"code","execution_count":null,"id":"796ab80b","metadata":{"id":"796ab80b"},"outputs":[],"source":["sc.pp.scale(adata, zero_center=False)"]},{"cell_type":"markdown","id":"3dce232c","metadata":{"id":"3dce232c"},"source":["# Performing the PCA and kNN analysis"]},{"cell_type":"code","execution_count":null,"id":"02f82eae","metadata":{"id":"02f82eae"},"outputs":[],"source":["sc.tl.pca(adata, svd_solver='arpack', n_comps = 150)"]},{"cell_type":"code","execution_count":null,"id":"ca7f59a8","metadata":{"id":"ca7f59a8"},"outputs":[],"source":["sc.pl.pca_variance_ratio(adata, n_pcs=150, log=True, save = True)"]},{"cell_type":"code","execution_count":null,"id":"c000970f","metadata":{"id":"c000970f"},"outputs":[],"source":["sc.pp.neighbors(adata, n_neighbors=45, n_pcs=105)"]},{"cell_type":"code","execution_count":null,"id":"04e3a45d","metadata":{"id":"04e3a45d"},"outputs":[],"source":["sc.tl.umap(adata, min_dist=0.5, spread = 1, alpha = 1, gamma = 1.0)"]},{"cell_type":"code","execution_count":null,"id":"26116f26","metadata":{"id":"26116f26"},"outputs":[],"source":["sc.pl.umap(adata)"]},{"cell_type":"code","execution_count":null,"id":"4b21c904","metadata":{"id":"4b21c904"},"outputs":[],"source":["tr = 'PrileiEVm023936t1'\n","sc.pl.umap(adata, color=tr, title = tr, color_map = umap_cmap, save = tr+'.pdf')"]},{"cell_type":"code","execution_count":null,"id":"2f4b7fda","metadata":{"id":"2f4b7fda"},"outputs":[],"source":["tr = 'PrileiEVm008309t1'\n","sc.pl.umap(adata, color=tr, title = tr, color_map = umap_cmap, save = tr+'.pdf')"]},{"cell_type":"code","execution_count":null,"id":"8ffe408d","metadata":{"id":"8ffe408d"},"outputs":[],"source":["tr = 'PrileiEVm011741t1'\n","sc.pl.umap(adata, color=tr, title = tr, color_map = umap_cmap, save = tr+'.pdf')"]},{"cell_type":"code","execution_count":null,"id":"247ab6bc","metadata":{"id":"247ab6bc"},"outputs":[],"source":["tr = 'PrileiEVm021316t1'\n","sc.pl.umap(adata, color=tr, title = tr, color_map = umap_cmap, save = tr+'.pdf')"]},{"cell_type":"code","execution_count":null,"id":"7658d5ff","metadata":{"id":"7658d5ff"},"outputs":[],"source":["tr = 'PrileiEVm022250t1'\n","sc.pl.umap(adata, color=tr, title = tr, color_map = umap_cmap, save = tr+'.pdf')"]},{"cell_type":"code","execution_count":null,"id":"cdcebfc5","metadata":{"id":"cdcebfc5"},"outputs":[],"source":["tr = 'PrileiEVm000325t1'\n","sc.pl.umap(adata, color=tr, title = tr, color_map = umap_cmap, save = tr+'.pdf')"]},{"cell_type":"code","execution_count":null,"id":"a9089ea5","metadata":{"id":"a9089ea5"},"outputs":[],"source":["tr = 'PrileiEVm013699t1'\n","sc.pl.umap(adata, color=tr, title = tr, color_map = umap_cmap, save = tr+'.pdf')"]},{"cell_type":"code","execution_count":null,"id":"4a340d3a","metadata":{"id":"4a340d3a"},"outputs":[],"source":["tr = 'PrileiEVm020595t1'\n","sc.pl.umap(adata, color=tr, title = tr, color_map = umap_cmap, save = tr+'.pdf')"]},{"cell_type":"code","execution_count":null,"id":"df6c05f6","metadata":{"id":"df6c05f6"},"outputs":[],"source":[]},{"cell_type":"markdown","id":"4bfb1da3","metadata":{"id":"4bfb1da3"},"source":["# Clustering"]},{"cell_type":"code","execution_count":null,"id":"72770b23","metadata":{"id":"72770b23"},"outputs":[],"source":["resolutions = [0.5, 1, 1.5, 2]"]},{"cell_type":"code","execution_count":null,"id":"b8b19cbb","metadata":{"id":"b8b19cbb"},"outputs":[],"source":["for i in resolutions:\n","    sc.tl.leiden(adata, resolution = i, key_added = 'leiden_'+str(i))\n","    sc.pl.umap(adata, color='leiden_'+str(i))"]},{"cell_type":"code","execution_count":null,"id":"802eb466","metadata":{"id":"802eb466"},"outputs":[],"source":["leiden_names = adata.obs.columns[adata.obs.columns.str.contains('leiden')].to_list()"]},{"cell_type":"code","execution_count":null,"id":"72a0f50a","metadata":{"id":"72a0f50a"},"outputs":[],"source":["leiden_names"]},{"cell_type":"code","execution_count":null,"id":"7f367e4f","metadata":{"id":"7f367e4f"},"outputs":[],"source":["sc.pl.umap(adata, color=leiden_names, legend_loc = 'on data', legend_fontsize = 10)"]},{"cell_type":"code","execution_count":null,"id":"159751a9","metadata":{"id":"159751a9"},"outputs":[],"source":["for leiden_i in leiden_names:\n","    with rc_context({'figure.figsize': (15, 15)}):\n","        sc.pl.umap(adata, color=leiden_i, legend_loc='on data', title=str(leiden_i), size = 50, frameon=False, \n","                   save = '_'+str(leiden_i))"]},{"cell_type":"code","execution_count":null,"id":"890f5d4f","metadata":{"id":"890f5d4f"},"outputs":[],"source":["for leiden_i in leiden_names:\n","    sc.tl.rank_genes_groups(adata, leiden_i, method='logreg', key_added = 'rank_genes_groups_logreg_'+str(leiden_i))\n","    sc.pl.rank_genes_groups(adata, key='rank_genes_groups_logreg_'+str(leiden_i), n_genes = 10, sharey = False)"]},{"cell_type":"code","execution_count":null,"id":"085bd7e8","metadata":{"id":"085bd7e8"},"outputs":[],"source":["for leiden_i in leiden_names:\n","    sc.tl.rank_genes_groups(adata, leiden_i, method='wilcoxon', key_added = 'rank_genes_groups_wilcox_'+str(leiden_i))\n","    sc.pl.rank_genes_groups(adata, key='rank_genes_groups_wilcox_'+str(leiden_i), n_genes = 10, sharey = False)"]},{"cell_type":"code","execution_count":null,"id":"774ff402","metadata":{"id":"774ff402"},"outputs":[],"source":["adata.write(results_file)"]},{"cell_type":"code","execution_count":null,"id":"137e7708","metadata":{"id":"137e7708"},"outputs":[],"source":[]},{"cell_type":"code","execution_count":null,"id":"17a42da7","metadata":{"id":"17a42da7"},"outputs":[],"source":[]}],"metadata":{"colab":{"provenance":[]},"kernelspec":{"display_name":"Python 3","language":"python","name":"python3"},"language_info":{"codemirror_mode":{"name":"ipython","version":3},"file_extension":".py","mimetype":"text/x-python","name":"python","nbconvert_exporter":"python","pygments_lexer":"ipython3","version":"3.8.8"}},"nbformat":4,"nbformat_minor":5}