Blattman_Ecoli_Trajectory_Inference_and_DEG.ipynb

{"nbformat":4,"nbformat_minor":0,"metadata":{"colab":{"name":"Blattman_Ecoli_Trajectory_Inference_and_DEG.ipynb","provenance":[],"collapsed_sections":["9sf92iBpZbu6","js_cT97ez60v","K4YHqwKtn9at","djj7x0EHjK08"]},"kernelspec":{"name":"python3","display_name":"Python 3"},"language_info":{"name":"python"}},"cells":[{"cell_type":"markdown","source":["# Setup"],"metadata":{"id":"9sf92iBpZbu6"}},{"cell_type":"code","execution_count":null,"metadata":{"colab":{"base_uri":"https://localhost:8080/"},"id":"rFZ2OarAmjcY","executionInfo":{"status":"ok","timestamp":1650231369201,"user_tz":240,"elapsed":48402,"user":{"displayName":"Jacob Reis Rosenfeld","userId":"16476830039630475050"}},"outputId":"cfb330aa-d5da-4366-f2fd-e40baf761fee"},"outputs":[{"output_type":"stream","name":"stdout","text":["Requirement already satisfied: matplotlib in /usr/local/lib/python3.7/dist-packages (3.5.1)\n","Requirement already satisfied: pyparsing>=2.2.1 in /usr/local/lib/python3.7/dist-packages (from matplotlib) (3.0.8)\n","Requirement already satisfied: cycler>=0.10 in /usr/local/lib/python3.7/dist-packages (from matplotlib) (0.11.0)\n","Requirement already satisfied: packaging>=20.0 in /usr/local/lib/python3.7/dist-packages (from matplotlib) (21.3)\n","Requirement already satisfied: numpy>=1.17 in /usr/local/lib/python3.7/dist-packages (from matplotlib) (1.21.5)\n","Requirement already satisfied: python-dateutil>=2.7 in /usr/local/lib/python3.7/dist-packages (from matplotlib) (2.8.2)\n","Requirement already satisfied: pillow>=6.2.0 in /usr/local/lib/python3.7/dist-packages (from matplotlib) (7.1.2)\n","Requirement already satisfied: fonttools>=4.22.0 in /usr/local/lib/python3.7/dist-packages (from matplotlib) (4.32.0)\n","Requirement already satisfied: kiwisolver>=1.0.1 in /usr/local/lib/python3.7/dist-packages (from matplotlib) (1.4.2)\n","Requirement already satisfied: typing-extensions in /usr/local/lib/python3.7/dist-packages (from kiwisolver>=1.0.1->matplotlib) (4.1.1)\n","Requirement already satisfied: six>=1.5 in /usr/local/lib/python3.7/dist-packages (from python-dateutil>=2.7->matplotlib) (1.15.0)\n","Requirement already satisfied: scanpy in /usr/local/lib/python3.7/dist-packages (1.9.1)\n","Requirement already satisfied: tqdm in /usr/local/lib/python3.7/dist-packages (from scanpy) (4.64.0)\n","Requirement already satisfied: anndata>=0.7.4 in /usr/local/lib/python3.7/dist-packages (from scanpy) (0.8.0)\n","Requirement already satisfied: h5py>=3 in /usr/local/lib/python3.7/dist-packages (from scanpy) (3.1.0)\n","Requirement already satisfied: numba>=0.41.0 in /usr/local/lib/python3.7/dist-packages (from scanpy) (0.51.2)\n","Requirement already satisfied: patsy in /usr/local/lib/python3.7/dist-packages (from scanpy) (0.5.2)\n","Requirement already satisfied: natsort in /usr/local/lib/python3.7/dist-packages (from scanpy) (5.5.0)\n","Requirement already satisfied: networkx>=2.3 in /usr/local/lib/python3.7/dist-packages (from scanpy) (2.6.3)\n","Requirement already satisfied: scikit-learn>=0.22 in /usr/local/lib/python3.7/dist-packages (from scanpy) (1.0.2)\n","Requirement already satisfied: matplotlib>=3.4 in /usr/local/lib/python3.7/dist-packages (from scanpy) (3.5.1)\n","Requirement already satisfied: session-info in /usr/local/lib/python3.7/dist-packages (from scanpy) (1.0.0)\n","Requirement already satisfied: packaging in /usr/local/lib/python3.7/dist-packages (from scanpy) (21.3)\n","Requirement already satisfied: scipy>=1.4 in /usr/local/lib/python3.7/dist-packages (from scanpy) (1.4.1)\n","Requirement already satisfied: importlib_metadata>=0.7 in /usr/local/lib/python3.7/dist-packages (from scanpy) (4.11.3)\n","Requirement already satisfied: seaborn in /usr/local/lib/python3.7/dist-packages (from scanpy) (0.11.2)\n","Requirement already satisfied: numpy>=1.17.0 in /usr/local/lib/python3.7/dist-packages (from scanpy) (1.21.5)\n","Requirement already satisfied: statsmodels>=0.10.0rc2 in /usr/local/lib/python3.7/dist-packages (from scanpy) (0.10.2)\n","Requirement already satisfied: umap-learn>=0.3.10 in /usr/local/lib/python3.7/dist-packages (from scanpy) (0.5.3)\n","Requirement already satisfied: pandas>=1.0 in /usr/local/lib/python3.7/dist-packages (from scanpy) (1.3.5)\n","Requirement already satisfied: joblib in /usr/local/lib/python3.7/dist-packages (from scanpy) (1.1.0)\n","Requirement already satisfied: typing_extensions in /usr/local/lib/python3.7/dist-packages (from anndata>=0.7.4->scanpy) (4.1.1)\n","Requirement already satisfied: cached-property in /usr/local/lib/python3.7/dist-packages (from h5py>=3->scanpy) (1.5.2)\n","Requirement already satisfied: zipp>=0.5 in /usr/local/lib/python3.7/dist-packages (from importlib_metadata>=0.7->scanpy) (3.8.0)\n","Requirement already satisfied: kiwisolver>=1.0.1 in /usr/local/lib/python3.7/dist-packages (from matplotlib>=3.4->scanpy) (1.4.2)\n","Requirement already satisfied: pillow>=6.2.0 in /usr/local/lib/python3.7/dist-packages (from matplotlib>=3.4->scanpy) (7.1.2)\n","Requirement already satisfied: pyparsing>=2.2.1 in /usr/local/lib/python3.7/dist-packages (from matplotlib>=3.4->scanpy) (3.0.8)\n","Requirement already satisfied: cycler>=0.10 in /usr/local/lib/python3.7/dist-packages (from matplotlib>=3.4->scanpy) (0.11.0)\n","Requirement already satisfied: fonttools>=4.22.0 in /usr/local/lib/python3.7/dist-packages (from matplotlib>=3.4->scanpy) (4.32.0)\n","Requirement already satisfied: python-dateutil>=2.7 in /usr/local/lib/python3.7/dist-packages (from matplotlib>=3.4->scanpy) (2.8.2)\n","Requirement already satisfied: llvmlite<0.35,>=0.34.0.dev0 in /usr/local/lib/python3.7/dist-packages (from numba>=0.41.0->scanpy) (0.34.0)\n","Requirement already satisfied: setuptools in /usr/local/lib/python3.7/dist-packages (from numba>=0.41.0->scanpy) (57.4.0)\n","Requirement already satisfied: pytz>=2017.3 in /usr/local/lib/python3.7/dist-packages (from pandas>=1.0->scanpy) (2018.9)\n","Requirement already satisfied: six>=1.5 in /usr/local/lib/python3.7/dist-packages (from python-dateutil>=2.7->matplotlib>=3.4->scanpy) (1.15.0)\n","Requirement already satisfied: threadpoolctl>=2.0.0 in /usr/local/lib/python3.7/dist-packages (from scikit-learn>=0.22->scanpy) (3.1.0)\n","Requirement already satisfied: pynndescent>=0.5 in /usr/local/lib/python3.7/dist-packages (from umap-learn>=0.3.10->scanpy) (0.5.6)\n","Requirement already satisfied: stdlib-list in /usr/local/lib/python3.7/dist-packages (from session-info->scanpy) (0.8.0)\n","Requirement already satisfied: numpy in /usr/local/lib/python3.7/dist-packages (1.21.5)\n","Requirement already satisfied: pandas in /usr/local/lib/python3.7/dist-packages (1.3.5)\n","Requirement already satisfied: numpy>=1.17.3 in /usr/local/lib/python3.7/dist-packages (from pandas) (1.21.5)\n","Requirement already satisfied: python-dateutil>=2.7.3 in /usr/local/lib/python3.7/dist-packages (from pandas) (2.8.2)\n","Requirement already satisfied: pytz>=2017.3 in /usr/local/lib/python3.7/dist-packages (from pandas) (2018.9)\n","Requirement already satisfied: six>=1.5 in /usr/local/lib/python3.7/dist-packages (from python-dateutil>=2.7.3->pandas) (1.15.0)\n","Requirement already satisfied: AnnData in /usr/local/lib/python3.7/dist-packages (0.8.0)\n","Requirement already satisfied: natsort in /usr/local/lib/python3.7/dist-packages (from AnnData) (5.5.0)\n","Requirement already satisfied: packaging>=20 in /usr/local/lib/python3.7/dist-packages (from AnnData) (21.3)\n","Requirement already satisfied: scipy>1.4 in /usr/local/lib/python3.7/dist-packages (from AnnData) (1.4.1)\n","Requirement already satisfied: pandas>=1.1.1 in /usr/local/lib/python3.7/dist-packages (from AnnData) (1.3.5)\n","Requirement already satisfied: typing_extensions in /usr/local/lib/python3.7/dist-packages (from AnnData) (4.1.1)\n","Requirement already satisfied: importlib_metadata>=0.7 in /usr/local/lib/python3.7/dist-packages (from AnnData) (4.11.3)\n","Requirement already satisfied: h5py>=3 in /usr/local/lib/python3.7/dist-packages (from AnnData) (3.1.0)\n","Requirement already satisfied: numpy>=1.16.5 in /usr/local/lib/python3.7/dist-packages (from AnnData) (1.21.5)\n","Requirement already satisfied: cached-property in /usr/local/lib/python3.7/dist-packages (from h5py>=3->AnnData) (1.5.2)\n","Requirement already satisfied: zipp>=0.5 in /usr/local/lib/python3.7/dist-packages (from importlib_metadata>=0.7->AnnData) (3.8.0)\n","Requirement already satisfied: pyparsing!=3.0.5,>=2.0.2 in /usr/local/lib/python3.7/dist-packages (from packaging>=20->AnnData) (3.0.8)\n","Requirement already satisfied: pytz>=2017.3 in /usr/local/lib/python3.7/dist-packages (from pandas>=1.1.1->AnnData) (2018.9)\n","Requirement already satisfied: python-dateutil>=2.7.3 in /usr/local/lib/python3.7/dist-packages (from pandas>=1.1.1->AnnData) (2.8.2)\n","Requirement already satisfied: six>=1.5 in /usr/local/lib/python3.7/dist-packages (from python-dateutil>=2.7.3->pandas>=1.1.1->AnnData) (1.15.0)\n","Requirement already satisfied: wishbone_dev in /usr/local/lib/python3.7/dist-packages (0.5.2)\n","Requirement already satisfied: statsmodels>=0.8.0 in /usr/local/lib/python3.7/dist-packages (from wishbone_dev) (0.10.2)\n","Requirement already satisfied: seaborn>=0.7.1 in /usr/local/lib/python3.7/dist-packages (from wishbone_dev) (0.11.2)\n","Requirement already satisfied: matplotlib>=2.0.0 in /usr/local/lib/python3.7/dist-packages (from wishbone_dev) (3.5.1)\n","Requirement already satisfied: pandas>=0.19.2 in /usr/local/lib/python3.7/dist-packages (from wishbone_dev) (1.3.5)\n","Requirement already satisfied: bhtsne in /usr/local/lib/python3.7/dist-packages (from wishbone_dev) (0.1.9)\n","Requirement already satisfied: numpy>=1.12.0 in /usr/local/lib/python3.7/dist-packages (from wishbone_dev) (1.21.5)\n","Requirement already satisfied: networkx>=1.11 in /usr/local/lib/python3.7/dist-packages (from wishbone_dev) (2.6.3)\n","Requirement already satisfied: fcsparser>=0.1.2 in /usr/local/lib/python3.7/dist-packages (from wishbone_dev) (0.2.4)\n","Requirement already satisfied: sklearn in /usr/local/lib/python3.7/dist-packages (from wishbone_dev) (0.0)\n","Requirement already satisfied: Cython in /usr/local/lib/python3.7/dist-packages (from wishbone_dev) (0.29.28)\n","Requirement already satisfied: scipy>=0.18.1 in /usr/local/lib/python3.7/dist-packages (from wishbone_dev) (1.4.1)\n","Requirement already satisfied: six in /usr/local/lib/python3.7/dist-packages (from fcsparser>=0.1.2->wishbone_dev) (1.15.0)\n","Requirement already satisfied: setuptools in /usr/local/lib/python3.7/dist-packages (from fcsparser>=0.1.2->wishbone_dev) (57.4.0)\n","Requirement already satisfied: fonttools>=4.22.0 in /usr/local/lib/python3.7/dist-packages (from matplotlib>=2.0.0->wishbone_dev) (4.32.0)\n","Requirement already satisfied: packaging>=20.0 in /usr/local/lib/python3.7/dist-packages (from matplotlib>=2.0.0->wishbone_dev) (21.3)\n","Requirement already satisfied: pyparsing>=2.2.1 in /usr/local/lib/python3.7/dist-packages (from matplotlib>=2.0.0->wishbone_dev) (3.0.8)\n","Requirement already satisfied: kiwisolver>=1.0.1 in /usr/local/lib/python3.7/dist-packages (from matplotlib>=2.0.0->wishbone_dev) (1.4.2)\n","Requirement already satisfied: python-dateutil>=2.7 in /usr/local/lib/python3.7/dist-packages (from matplotlib>=2.0.0->wishbone_dev) (2.8.2)\n","Requirement already satisfied: pillow>=6.2.0 in /usr/local/lib/python3.7/dist-packages (from matplotlib>=2.0.0->wishbone_dev) (7.1.2)\n","Requirement already satisfied: cycler>=0.10 in /usr/local/lib/python3.7/dist-packages (from matplotlib>=2.0.0->wishbone_dev) (0.11.0)\n","Requirement already satisfied: typing-extensions in /usr/local/lib/python3.7/dist-packages (from kiwisolver>=1.0.1->matplotlib>=2.0.0->wishbone_dev) (4.1.1)\n","Requirement already satisfied: pytz>=2017.3 in /usr/local/lib/python3.7/dist-packages (from pandas>=0.19.2->wishbone_dev) (2018.9)\n","Requirement already satisfied: patsy>=0.4.0 in /usr/local/lib/python3.7/dist-packages (from statsmodels>=0.8.0->wishbone_dev) (0.5.2)\n","Requirement already satisfied: scikit-learn in /usr/local/lib/python3.7/dist-packages (from sklearn->wishbone_dev) (1.0.2)\n","Requirement already satisfied: joblib>=0.11 in /usr/local/lib/python3.7/dist-packages (from scikit-learn->sklearn->wishbone_dev) (1.1.0)\n","Requirement already satisfied: threadpoolctl>=2.0.0 in /usr/local/lib/python3.7/dist-packages (from scikit-learn->sklearn->wishbone_dev) (3.1.0)\n","Requirement already satisfied: leidenalg in /usr/local/lib/python3.7/dist-packages (0.8.9)\n","Requirement already satisfied: igraph<0.10,>=0.9.0 in /usr/local/lib/python3.7/dist-packages (from leidenalg) (0.9.10)\n","Requirement already satisfied: texttable>=1.6.2 in /usr/local/lib/python3.7/dist-packages (from igraph<0.10,>=0.9.0->leidenalg) (1.6.4)\n"]}],"source":["!pip install -U matplotlib\n","!pip install scanpy\n","!pip install numpy\n","!pip install pandas\n","!pip install AnnData\n","!pip install wishbone_dev\n","!pip install leidenalg\n","\n","import scanpy as sc\n","import numpy as np\n","import pandas as pd\n","import matplotlib.pyplot as plt\n","import anndata as ad"]},{"cell_type":"markdown","source":["# Blattman E.coli data preprocessing"],"metadata":{"id":"js_cT97ez60v"}},{"cell_type":"code","source":["blattman_ecoli = sc.read('/Blattman data/GSM4192509_394A_R2_trimmed_v4_v11_threshold_1_unfiltered_gene_matrix.txt', delimiter = '\\t')\n","blattman_ecoli"],"metadata":{"colab":{"base_uri":"https://localhost:8080/"},"id":"fiCvAFaHyXMz","executionInfo":{"status":"ok","timestamp":1650231396227,"user_tz":240,"elapsed":25839,"user":{"displayName":"Jacob Reis Rosenfeld","userId":"16476830039630475050"}},"outputId":"4acc2d6f-df77-4ff7-d1c5-5526b66fd790"},"execution_count":null,"outputs":[{"output_type":"execute_result","data":{"text/plain":["AnnData object with n_obs × n_vars = 29548 × 2423"]},"metadata":{},"execution_count":3}]},{"cell_type":"code","execution_count":null,"metadata":{"colab":{"base_uri":"https://localhost:8080/"},"id":"29982c05","executionInfo":{"status":"ok","timestamp":1650231398858,"user_tz":240,"elapsed":2636,"user":{"displayName":"Jacob Reis Rosenfeld","userId":"16476830039630475050"}},"outputId":"5e8c3c6f-c726-4e1d-9637-747cc3681c0f"},"outputs":[{"output_type":"stream","name":"stderr","text":["/usr/local/lib/python3.7/dist-packages/scanpy/preprocessing/_qc.py:422: RuntimeWarning: invalid value encountered in true_divide\n","  return values / sums[:, None]\n"]}],"source":["sc.pp.calculate_qc_metrics(blattman_ecoli, inplace= True)"]},{"cell_type":"code","execution_count":null,"metadata":{"id":"eed64d2d"},"outputs":[],"source":["dense2 = blattman_ecoli.copy()"]},{"cell_type":"code","execution_count":null,"metadata":{"id":"b4a11980","executionInfo":{"status":"ok","timestamp":1650231400571,"user_tz":240,"elapsed":1288,"user":{"displayName":"Jacob Reis Rosenfeld","userId":"16476830039630475050"}},"colab":{"base_uri":"https://localhost:8080/"},"outputId":"d04d093d-ef70-4e37-f130-47ca3efcd3f0"},"outputs":[{"output_type":"stream","name":"stderr","text":["/usr/local/lib/python3.7/dist-packages/scanpy/preprocessing/_simple.py:138: ImplicitModificationWarning: Trying to modify attribute `.obs` of view, initializing view as actual.\n","  adata.obs['n_counts'] = number\n"]}],"source":["dense2 = dense2[:,:2418]\n","sc.pp.filter_cells(dense2,min_counts=15)\n","sc.pp.filter_cells(dense2,max_counts = 2000)\n","sc.pp.filter_genes(dense2,min_cells = 5)"]},{"cell_type":"code","execution_count":null,"metadata":{"colab":{"base_uri":"https://localhost:8080/"},"id":"e7a6e37f","executionInfo":{"status":"ok","timestamp":1650231400572,"user_tz":240,"elapsed":9,"user":{"displayName":"Jacob Reis Rosenfeld","userId":"16476830039630475050"}},"outputId":"c50f8df8-142b-40bc-a348-214bbf139ee1"},"outputs":[{"output_type":"execute_result","data":{"text/plain":["AnnData object with n_obs × n_vars = 12255 × 1983\n","    obs: 'n_genes_by_counts', 'log1p_n_genes_by_counts', 'total_counts', 'log1p_total_counts', 'pct_counts_in_top_50_genes', 'pct_counts_in_top_100_genes', 'pct_counts_in_top_200_genes', 'pct_counts_in_top_500_genes', 'n_counts'\n","    var: 'n_cells_by_counts', 'mean_counts', 'log1p_mean_counts', 'pct_dropout_by_counts', 'total_counts', 'log1p_total_counts', 'n_cells'"]},"metadata":{},"execution_count":7}],"source":["dense2"]},{"cell_type":"code","execution_count":null,"metadata":{"colab":{"base_uri":"https://localhost:8080/"},"id":"f13c44c0","executionInfo":{"status":"ok","timestamp":1650231400573,"user_tz":240,"elapsed":7,"user":{"displayName":"Jacob Reis Rosenfeld","userId":"16476830039630475050"}},"outputId":"51d83eb6-c927-47d7-b902-d9d8356d6b7e"},"outputs":[{"output_type":"execute_result","data":{"text/plain":["394A_bc1_10_bc2_10_bc3_79_none_0       201.000031\n","394A_bc1_10_bc2_11_bc3_7_GFP_1         200.999985\n","394A_bc1_10_bc2_12_bc3_83_GFP_3        201.000015\n","394A_bc1_10_bc2_15_bc3_43_none_5       201.000000\n","394A_bc1_10_bc2_15_bc3_7_GFP_6         201.000000\n","                                          ...    \n","394A_bc1_9_bc2_95_bc3_37_none_29540    201.000000\n","394A_bc1_9_bc2_95_bc3_43_GFP_29541     201.000031\n","394A_bc1_9_bc2_9_bc3_36_RFP_29544      201.000000\n","394A_bc1_9_bc2_9_bc3_50_GFP_29545      201.000031\n","394A_bc1_9_bc2_9_bc3_84_none_29547     200.999985\n","Name: cell_sum, Length: 12255, dtype: float32"]},"metadata":{},"execution_count":8}],"source":["sc.pp.normalize_total(dense2, inplace = True)\n","dense2.obs['cell_sum']=dense2.X.sum(axis=1)\n","dense2.obs['cell_sum']"]},{"cell_type":"code","execution_count":null,"metadata":{"id":"202ca8d8"},"outputs":[],"source":["sc.pp.log1p(dense2)"]},{"cell_type":"code","execution_count":null,"metadata":{"id":"ca02a041"},"outputs":[],"source":["#sc.pp.regress_out(dense2,['total_counts'])\n","sc.pp.scale(dense2,max_value=10)"]},{"cell_type":"code","execution_count":null,"metadata":{"colab":{"base_uri":"https://localhost:8080/","height":487},"id":"cf6931c4","executionInfo":{"status":"ok","timestamp":1650231401464,"user_tz":240,"elapsed":8,"user":{"displayName":"Jacob Reis Rosenfeld","userId":"16476830039630475050"}},"outputId":"aac64d1d-3ca2-4dab-fa43-3c28901194c1"},"outputs":[{"output_type":"execute_result","data":{"text/plain":["                                     n_genes_by_counts  \\\n","394A_bc1_10_bc2_10_bc3_79_none_0                    28   \n","394A_bc1_10_bc2_11_bc3_7_GFP_1                      40   \n","394A_bc1_10_bc2_12_bc3_83_GFP_3                     71   \n","394A_bc1_10_bc2_15_bc3_43_none_5                     6   \n","394A_bc1_10_bc2_15_bc3_7_GFP_6                      43   \n","...                                                ...   \n","394A_bc1_9_bc2_95_bc3_37_none_29540                 31   \n","394A_bc1_9_bc2_95_bc3_43_GFP_29541                  26   \n","394A_bc1_9_bc2_9_bc3_36_RFP_29544                   50   \n","394A_bc1_9_bc2_9_bc3_50_GFP_29545                   74   \n","394A_bc1_9_bc2_9_bc3_84_none_29547                  60   \n","\n","                                     log1p_n_genes_by_counts  total_counts  \\\n","394A_bc1_10_bc2_10_bc3_79_none_0                    3.367296         202.0   \n","394A_bc1_10_bc2_11_bc3_7_GFP_1                      3.713572         431.0   \n","394A_bc1_10_bc2_12_bc3_83_GFP_3                     4.276666         755.0   \n","394A_bc1_10_bc2_15_bc3_43_none_5                    1.945910          25.0   \n","394A_bc1_10_bc2_15_bc3_7_GFP_6                      3.784190         469.0   \n","...                                                      ...           ...   \n","394A_bc1_9_bc2_95_bc3_37_none_29540                 3.465736         232.0   \n","394A_bc1_9_bc2_95_bc3_43_GFP_29541                  3.295837         355.0   \n","394A_bc1_9_bc2_9_bc3_36_RFP_29544                   3.931826         454.0   \n","394A_bc1_9_bc2_9_bc3_50_GFP_29545                   4.317488         971.0   \n","394A_bc1_9_bc2_9_bc3_84_none_29547                  4.110874         601.0   \n","\n","                                     log1p_total_counts  \\\n","394A_bc1_10_bc2_10_bc3_79_none_0               5.313206   \n","394A_bc1_10_bc2_11_bc3_7_GFP_1                 6.068426   \n","394A_bc1_10_bc2_12_bc3_83_GFP_3                6.628041   \n","394A_bc1_10_bc2_15_bc3_43_none_5               3.258096   \n","394A_bc1_10_bc2_15_bc3_7_GFP_6                 6.152733   \n","...                                                 ...   \n","394A_bc1_9_bc2_95_bc3_37_none_29540            5.451038   \n","394A_bc1_9_bc2_95_bc3_43_GFP_29541             5.874931   \n","394A_bc1_9_bc2_9_bc3_36_RFP_29544              6.120297   \n","394A_bc1_9_bc2_9_bc3_50_GFP_29545              6.879356   \n","394A_bc1_9_bc2_9_bc3_84_none_29547             6.400258   \n","\n","                                     pct_counts_in_top_50_genes  \\\n","394A_bc1_10_bc2_10_bc3_79_none_0                     100.000000   \n","394A_bc1_10_bc2_11_bc3_7_GFP_1                       100.000000   \n","394A_bc1_10_bc2_12_bc3_83_GFP_3                       97.218543   \n","394A_bc1_10_bc2_15_bc3_43_none_5                     100.000000   \n","394A_bc1_10_bc2_15_bc3_7_GFP_6                       100.000000   \n","...                                                         ...   \n","394A_bc1_9_bc2_95_bc3_37_none_29540                  100.000000   \n","394A_bc1_9_bc2_95_bc3_43_GFP_29541                   100.000000   \n","394A_bc1_9_bc2_9_bc3_36_RFP_29544                    100.000000   \n","394A_bc1_9_bc2_9_bc3_50_GFP_29545                     97.528321   \n","394A_bc1_9_bc2_9_bc3_84_none_29547                    98.336106   \n","\n","                                     pct_counts_in_top_100_genes  \\\n","394A_bc1_10_bc2_10_bc3_79_none_0                           100.0   \n","394A_bc1_10_bc2_11_bc3_7_GFP_1                             100.0   \n","394A_bc1_10_bc2_12_bc3_83_GFP_3                            100.0   \n","394A_bc1_10_bc2_15_bc3_43_none_5                           100.0   \n","394A_bc1_10_bc2_15_bc3_7_GFP_6                             100.0   \n","...                                                          ...   \n","394A_bc1_9_bc2_95_bc3_37_none_29540                        100.0   \n","394A_bc1_9_bc2_95_bc3_43_GFP_29541                         100.0   \n","394A_bc1_9_bc2_9_bc3_36_RFP_29544                          100.0   \n","394A_bc1_9_bc2_9_bc3_50_GFP_29545                          100.0   \n","394A_bc1_9_bc2_9_bc3_84_none_29547                         100.0   \n","\n","                                     pct_counts_in_top_200_genes  \\\n","394A_bc1_10_bc2_10_bc3_79_none_0                           100.0   \n","394A_bc1_10_bc2_11_bc3_7_GFP_1                             100.0   \n","394A_bc1_10_bc2_12_bc3_83_GFP_3                            100.0   \n","394A_bc1_10_bc2_15_bc3_43_none_5                           100.0   \n","394A_bc1_10_bc2_15_bc3_7_GFP_6                             100.0   \n","...                                                          ...   \n","394A_bc1_9_bc2_95_bc3_37_none_29540                        100.0   \n","394A_bc1_9_bc2_95_bc3_43_GFP_29541                         100.0   \n","394A_bc1_9_bc2_9_bc3_36_RFP_29544                          100.0   \n","394A_bc1_9_bc2_9_bc3_50_GFP_29545                          100.0   \n","394A_bc1_9_bc2_9_bc3_84_none_29547                         100.0   \n","\n","                                     pct_counts_in_top_500_genes  n_counts  \\\n","394A_bc1_10_bc2_10_bc3_79_none_0                           100.0     202.0   \n","394A_bc1_10_bc2_11_bc3_7_GFP_1                             100.0     429.0   \n","394A_bc1_10_bc2_12_bc3_83_GFP_3                            100.0     747.0   \n","394A_bc1_10_bc2_15_bc3_43_none_5                           100.0      25.0   \n","394A_bc1_10_bc2_15_bc3_7_GFP_6                             100.0     468.0   \n","...                                                          ...       ...   \n","394A_bc1_9_bc2_95_bc3_37_none_29540                        100.0     232.0   \n","394A_bc1_9_bc2_95_bc3_43_GFP_29541                         100.0     353.0   \n","394A_bc1_9_bc2_9_bc3_36_RFP_29544                          100.0     453.0   \n","394A_bc1_9_bc2_9_bc3_50_GFP_29545                          100.0     970.0   \n","394A_bc1_9_bc2_9_bc3_84_none_29547                         100.0     601.0   \n","\n","                                       cell_sum express  \n","394A_bc1_10_bc2_10_bc3_79_none_0     201.000031    none  \n","394A_bc1_10_bc2_11_bc3_7_GFP_1       200.999985     GFP  \n","394A_bc1_10_bc2_12_bc3_83_GFP_3      201.000015     GFP  \n","394A_bc1_10_bc2_15_bc3_43_none_5     201.000000    none  \n","394A_bc1_10_bc2_15_bc3_7_GFP_6       201.000000     GFP  \n","...                                         ...     ...  \n","394A_bc1_9_bc2_95_bc3_37_none_29540  201.000000    none  \n","394A_bc1_9_bc2_95_bc3_43_GFP_29541   201.000031     GFP  \n","394A_bc1_9_bc2_9_bc3_36_RFP_29544    201.000000     RFP  \n","394A_bc1_9_bc2_9_bc3_50_GFP_29545    201.000031     GFP  \n","394A_bc1_9_bc2_9_bc3_84_none_29547   200.999985    none  \n","\n","[12255 rows x 11 columns]"],"text/html":["\n","  <div id=\"df-731f425d-9e7c-402f-b3d4-2c7ce645c6a9\">\n","    <div class=\"colab-df-container\">\n","      <div>\n","<style scoped>\n","    .dataframe tbody tr th:only-of-type {\n","        vertical-align: middle;\n","    }\n","\n","    .dataframe tbody tr th {\n","        vertical-align: top;\n","    }\n","\n","    .dataframe thead th {\n","        text-align: right;\n","    }\n","</style>\n","<table border=\"1\" class=\"dataframe\">\n","  <thead>\n","    <tr style=\"text-align: right;\">\n","      <th></th>\n","      <th>n_genes_by_counts</th>\n","      <th>log1p_n_genes_by_counts</th>\n","      <th>total_counts</th>\n","      <th>log1p_total_counts</th>\n","      <th>pct_counts_in_top_50_genes</th>\n","      <th>pct_counts_in_top_100_genes</th>\n","      <th>pct_counts_in_top_200_genes</th>\n","      <th>pct_counts_in_top_500_genes</th>\n","      <th>n_counts</th>\n","      <th>cell_sum</th>\n","      <th>express</th>\n","    </tr>\n","  </thead>\n","  <tbody>\n","    <tr>\n","      <th>394A_bc1_10_bc2_10_bc3_79_none_0</th>\n","      <td>28</td>\n","      <td>3.367296</td>\n","      <td>202.0</td>\n","      <td>5.313206</td>\n","      <td>100.000000</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>202.0</td>\n","      <td>201.000031</td>\n","      <td>none</td>\n","    </tr>\n","    <tr>\n","      <th>394A_bc1_10_bc2_11_bc3_7_GFP_1</th>\n","      <td>40</td>\n","      <td>3.713572</td>\n","      <td>431.0</td>\n","      <td>6.068426</td>\n","      <td>100.000000</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>429.0</td>\n","      <td>200.999985</td>\n","      <td>GFP</td>\n","    </tr>\n","    <tr>\n","      <th>394A_bc1_10_bc2_12_bc3_83_GFP_3</th>\n","      <td>71</td>\n","      <td>4.276666</td>\n","      <td>755.0</td>\n","      <td>6.628041</td>\n","      <td>97.218543</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>747.0</td>\n","      <td>201.000015</td>\n","      <td>GFP</td>\n","    </tr>\n","    <tr>\n","      <th>394A_bc1_10_bc2_15_bc3_43_none_5</th>\n","      <td>6</td>\n","      <td>1.945910</td>\n","      <td>25.0</td>\n","      <td>3.258096</td>\n","      <td>100.000000</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>25.0</td>\n","      <td>201.000000</td>\n","      <td>none</td>\n","    </tr>\n","    <tr>\n","      <th>394A_bc1_10_bc2_15_bc3_7_GFP_6</th>\n","      <td>43</td>\n","      <td>3.784190</td>\n","      <td>469.0</td>\n","      <td>6.152733</td>\n","      <td>100.000000</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>468.0</td>\n","      <td>201.000000</td>\n","      <td>GFP</td>\n","    </tr>\n","    <tr>\n","      <th>...</th>\n","      <td>...</td>\n","      <td>...</td>\n","      <td>...</td>\n","      <td>...</td>\n","      <td>...</td>\n","      <td>...</td>\n","      <td>...</td>\n","      <td>...</td>\n","      <td>...</td>\n","      <td>...</td>\n","      <td>...</td>\n","    </tr>\n","    <tr>\n","      <th>394A_bc1_9_bc2_95_bc3_37_none_29540</th>\n","      <td>31</td>\n","      <td>3.465736</td>\n","      <td>232.0</td>\n","      <td>5.451038</td>\n","      <td>100.000000</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>232.0</td>\n","      <td>201.000000</td>\n","      <td>none</td>\n","    </tr>\n","    <tr>\n","      <th>394A_bc1_9_bc2_95_bc3_43_GFP_29541</th>\n","      <td>26</td>\n","      <td>3.295837</td>\n","      <td>355.0</td>\n","      <td>5.874931</td>\n","      <td>100.000000</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>353.0</td>\n","      <td>201.000031</td>\n","      <td>GFP</td>\n","    </tr>\n","    <tr>\n","      <th>394A_bc1_9_bc2_9_bc3_36_RFP_29544</th>\n","      <td>50</td>\n","      <td>3.931826</td>\n","      <td>454.0</td>\n","      <td>6.120297</td>\n","      <td>100.000000</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>453.0</td>\n","      <td>201.000000</td>\n","      <td>RFP</td>\n","    </tr>\n","    <tr>\n","      <th>394A_bc1_9_bc2_9_bc3_50_GFP_29545</th>\n","      <td>74</td>\n","      <td>4.317488</td>\n","      <td>971.0</td>\n","      <td>6.879356</td>\n","      <td>97.528321</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>970.0</td>\n","      <td>201.000031</td>\n","      <td>GFP</td>\n","    </tr>\n","    <tr>\n","      <th>394A_bc1_9_bc2_9_bc3_84_none_29547</th>\n","      <td>60</td>\n","      <td>4.110874</td>\n","      <td>601.0</td>\n","      <td>6.400258</td>\n","      <td>98.336106</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>100.0</td>\n","      <td>601.0</td>\n","      <td>200.999985</td>\n","      <td>none</td>\n","    </tr>\n","  </tbody>\n","</table>\n","<p>12255 rows × 11 columns</p>\n","</div>\n","      <button class=\"colab-df-convert\" onclick=\"convertToInteractive('df-731f425d-9e7c-402f-b3d4-2c7ce645c6a9')\"\n","              title=\"Convert this dataframe to an interactive table.\"\n","              style=\"display:none;\">\n","        \n","  <svg xmlns=\"http://www.w3.org/2000/svg\" height=\"24px\"viewBox=\"0 0 24 24\"\n","       width=\"24px\">\n","    <path d=\"M0 0h24v24H0V0z\" fill=\"none\"/>\n","    <path d=\"M18.56 5.44l.94 2.06.94-2.06 2.06-.94-2.06-.94-.94-2.06-.94 2.06-2.06.94zm-11 1L8.5 8.5l.94-2.06 2.06-.94-2.06-.94L8.5 2.5l-.94 2.06-2.06.94zm10 10l.94 2.06.94-2.06 2.06-.94-2.06-.94-.94-2.06-.94 2.06-2.06.94z\"/><path d=\"M17.41 7.96l-1.37-1.37c-.4-.4-.92-.59-1.43-.59-.52 0-1.04.2-1.43.59L10.3 9.45l-7.72 7.72c-.78.78-.78 2.05 0 2.83L4 21.41c.39.39.9.59 1.41.59.51 0 1.02-.2 1.41-.59l7.78-7.78 2.81-2.81c.8-.78.8-2.07 0-2.86zM5.41 20L4 18.59l7.72-7.72 1.47 1.35L5.41 20z\"/>\n","  </svg>\n","      </button>\n","      \n","  <style>\n","    .colab-df-container {\n","      display:flex;\n","      flex-wrap:wrap;\n","      gap: 12px;\n","    }\n","\n","    .colab-df-convert {\n","      background-color: #E8F0FE;\n","      border: none;\n","      border-radius: 50%;\n","      cursor: pointer;\n","      display: none;\n","      fill: #1967D2;\n","      height: 32px;\n","      padding: 0 0 0 0;\n","      width: 32px;\n","    }\n","\n","    .colab-df-convert:hover {\n","      background-color: #E2EBFA;\n","      box-shadow: 0px 1px 2px rgba(60, 64, 67, 0.3), 0px 1px 3px 1px rgba(60, 64, 67, 0.15);\n","      fill: #174EA6;\n","    }\n","\n","    [theme=dark] .colab-df-convert {\n","      background-color: #3B4455;\n","      fill: #D2E3FC;\n","    }\n","\n","    [theme=dark] .colab-df-convert:hover {\n","      background-color: #434B5C;\n","      box-shadow: 0px 1px 3px 1px rgba(0, 0, 0, 0.15);\n","      filter: drop-shadow(0px 1px 2px rgba(0, 0, 0, 0.3));\n","      fill: #FFFFFF;\n","    }\n","  </style>\n","\n","      <script>\n","        const buttonEl =\n","          document.querySelector('#df-731f425d-9e7c-402f-b3d4-2c7ce645c6a9 button.colab-df-convert');\n","        buttonEl.style.display =\n","          google.colab.kernel.accessAllowed ? 'block' : 'none';\n","\n","        async function convertToInteractive(key) {\n","          const element = document.querySelector('#df-731f425d-9e7c-402f-b3d4-2c7ce645c6a9');\n","          const dataTable =\n","            await google.colab.kernel.invokeFunction('convertToInteractive',\n","                                                     [key], {});\n","          if (!dataTable) return;\n","\n","          const docLinkHtml = 'Like what you see? Visit the ' +\n","            '<a target=\"_blank\" href=https://colab.research.google.com/notebooks/data_table.ipynb>data table notebook</a>'\n","            + ' to learn more about interactive tables.';\n","          element.innerHTML = '';\n","          dataTable['output_type'] = 'display_data';\n","          await google.colab.output.renderOutput(dataTable, element);\n","          const docLink = document.createElement('div');\n","          docLink.innerHTML = docLinkHtml;\n","          element.appendChild(docLink);\n","        }\n","      </script>\n","    </div>\n","  </div>\n","  "]},"metadata":{},"execution_count":11}],"source":["express = []\n","for i in dense2.obs_names:\n","    if 'none' in i:\n","        express.append('none')\n","    elif 'RFP' in i:\n","        express.append('RFP')\n","    elif 'GFP' in i:\n","        express.append('GFP')\n","dense2.obs['express']= express\n","dense2.obs  "]},{"cell_type":"code","source":["import scipy\n","\n","sc.pp.pca(dense2, n_comps = 100)\n","sc.pp.neighbors(dense2, n_pcs = 100, n_neighbors = 15)"],"metadata":{"colab":{"base_uri":"https://localhost:8080/"},"id":"LZ_7opcvpCw6","executionInfo":{"status":"ok","timestamp":1650231448746,"user_tz":240,"elapsed":47287,"user":{"displayName":"Jacob Reis Rosenfeld","userId":"16476830039630475050"}},"outputId":"a0d8e410-09f2-49c1-d7e4-26720feca717"},"execution_count":null,"outputs":[{"output_type":"stream","name":"stderr","text":["/usr/local/lib/python3.7/dist-packages/numba/np/ufunc/parallel.py:363: NumbaWarning: The TBB threading layer requires TBB version 2019.5 or later i.e., TBB_INTERFACE_VERSION >= 11005. Found TBB_INTERFACE_VERSION = 9107. The TBB threading layer is disabled.\n","  warnings.warn(problem)\n"]}]},{"cell_type":"code","source":[""],"metadata":{"id":"RrijSHvt4JJH"},"execution_count":null,"outputs":[]},{"cell_type":"code","source":[""],"metadata":{"id":"xCKzLrmZpwVv"},"execution_count":null,"outputs":[]},{"cell_type":"markdown","source":["# Wishbone Starting Cell Identification and Trajectory"],"metadata":{"id":"K4YHqwKtn9at"}},{"cell_type":"markdown","source":["Runs Wishbone algorithm, separated anaylsis and running the algorithm in due to matplotlib issues"],"metadata":{"id":"JJqqReIRT26_"}},{"cell_type":"code","source":["import scanpy.external as sce\n","\n","ecoli_blattman_copy = dense2.copy()\n","id = ecoli_blattman_copy.obs[:]['n_genes_by_counts'].idxmax()\n","print(id)"],"metadata":{"id":"c_dPHm3RoEtK","executionInfo":{"status":"ok","timestamp":1650231448748,"user_tz":240,"elapsed":11,"user":{"displayName":"Jacob Reis Rosenfeld","userId":"16476830039630475050"}},"colab":{"base_uri":"https://localhost:8080/"},"outputId":"49ac2867-212e-4737-c7e7-4d3a29ef8f30"},"execution_count":null,"outputs":[{"output_type":"stream","name":"stdout","text":["394A_bc1_45_bc2_41_bc3_84_RFP_12773\n"]}]},{"cell_type":"code","source":["ecoli_blattman_copy"],"metadata":{"colab":{"base_uri":"https://localhost:8080/"},"id":"jLxpfQjx4tu7","executionInfo":{"status":"ok","timestamp":1650231448749,"user_tz":240,"elapsed":10,"user":{"displayName":"Jacob Reis Rosenfeld","userId":"16476830039630475050"}},"outputId":"664825b1-6cdf-4255-8eb2-390759973641"},"execution_count":null,"outputs":[{"output_type":"execute_result","data":{"text/plain":["AnnData object with n_obs × n_vars = 12255 × 1983\n","    obs: 'n_genes_by_counts', 'log1p_n_genes_by_counts', 'total_counts', 'log1p_total_counts', 'pct_counts_in_top_50_genes', 'pct_counts_in_top_100_genes', 'pct_counts_in_top_200_genes', 'pct_counts_in_top_500_genes', 'n_counts', 'cell_sum', 'express'\n","    var: 'n_cells_by_counts', 'mean_counts', 'log1p_mean_counts', 'pct_dropout_by_counts', 'total_counts', 'log1p_total_counts', 'n_cells', 'mean', 'std'\n","    uns: 'log1p', 'pca', 'neighbors'\n","    obsm: 'X_pca'\n","    varm: 'PCs'\n","    obsp: 'distances', 'connectivities'"]},"metadata":{},"execution_count":14}]},{"cell_type":"code","source":["sc.tl.tsne(adata=ecoli_blattman_copy, n_pcs=5, perplexity=30)"],"metadata":{"id":"1dPzro3WwKlZ","colab":{"base_uri":"https://localhost:8080/"},"executionInfo":{"status":"ok","timestamp":1650231582046,"user_tz":240,"elapsed":133304,"user":{"displayName":"Jacob Reis Rosenfeld","userId":"16476830039630475050"}},"outputId":"89d9b494-e204-40ae-a595-c1fcfa8d893d"},"execution_count":null,"outputs":[{"output_type":"stream","name":"stderr","text":["/usr/local/lib/python3.7/dist-packages/sklearn/manifold/_t_sne.py:783: FutureWarning: The default initialization in TSNE will change from 'random' to 'pca' in 1.2.\n","  FutureWarning,\n"]}]},{"cell_type":"code","source":["sc.pp.neighbors(adata=ecoli_blattman_copy) # for some reason adding on other parameters makes wishbone not work with key errors\n","sc.tl.diffmap(adata=ecoli_blattman_copy, n_comps=10)"],"metadata":{"id":"s0vkPhAVDumZ"},"execution_count":null,"outputs":[]},{"cell_type":"code","source":["state = True\n","while state:\n","  try:\n","    sce.tl.wishbone(adata=ecoli_blattman_copy, start_cell='394A_bc1_45_bc2_41_bc3_84_RFP_12773', components=[2, 3], num_waypoints=150, branch=True)\n","    state = False\n","  except KeyError:\n","    continue"],"metadata":{"colab":{"base_uri":"https://localhost:8080/"},"id":"1BNkzBeKuVBJ","executionInfo":{"status":"ok","timestamp":1650231744199,"user_tz":240,"elapsed":154378,"user":{"displayName":"Jacob Reis Rosenfeld","userId":"16476830039630475050"}},"outputId":"075dbff2-9f26-4138-adcd-04a643affa7b"},"execution_count":null,"outputs":[{"output_type":"stream","name":"stdout","text":["Building lNN graph...\n","lNN computed in : 0.06 seconds\n","Determining waypoints if not specified...\n","Determining shortest path distances and perspectives....\n","Building lNN graph...\n","lNN computed in : 0.06 seconds\n","Determining waypoints if not specified...\n","Determining shortest path distances and perspectives....\n","Building lNN graph...\n","lNN computed in : 0.06 seconds\n","Determining waypoints if not specified...\n","Determining shortest path distances and perspectives....\n","Building lNN graph...\n","lNN computed in : 0.06 seconds\n","Determining waypoints if not specified...\n","Determining shortest path distances and perspectives....\n","Building lNN graph...\n","lNN computed in : 0.06 seconds\n","Determining waypoints if not specified...\n","Determining shortest path distances and perspectives....\n","......................................................................................................................................................\n","Time for determining distances and perspectives: 117.00 seconds\n","Determining branch point and branch associations...\n","Running iterations...\n","Iteration: 2\n","Correlation with previous iteration:  0.9980\n","1 realignment iterations\n"]}]},{"cell_type":"code","source":["sc.tl.umap(ecoli_blattman_copy)"],"metadata":{"id":"tRIP4vXmvgXG"},"execution_count":null,"outputs":[]},{"cell_type":"code","source":[""],"metadata":{"id":"V6wniQjkTKZO"},"execution_count":null,"outputs":[]},{"cell_type":"code","source":["#sc.pl.tsne(ecoli_blattman_copy, color='trajectory_wishbone')"],"metadata":{"id":"EdsR0opBCxw-"},"execution_count":null,"outputs":[]},{"cell_type":"code","source":["#sc.pl.umap(ecoli_blattman_copy, color='branch_wishbone')"],"metadata":{"id":"TxmnF6Hv7eLo"},"execution_count":null,"outputs":[]},{"cell_type":"code","source":["#sc.pl.tsne(ecoli_blattman_copy, color='express')\n","#sc.pl.tsne(ecoli_blattman_copy, color=['zinT'])"],"metadata":{"id":"ofKiy1uYEsrQ"},"execution_count":null,"outputs":[]},{"cell_type":"markdown","source":["Above confirms Blattman findings that \"Of the 7,387 cells analysed, 61% did not contain any plasmid transcripts; their growth states were therefore ambiguous at first (grey points in PCA). However, using the PC1 threshold, we predicted that 92.2% of these were stationary-phase cells.Over-representation of stationary-phase cells in the ambiguous population was not surprising, as plasmid expression in stationary-phase cells was generally lower than in exponential-phase cells.\" \n","\n","also, trajectory moves from RFP to GFP"],"metadata":{"id":"jaao3RYCGQSj"}},{"cell_type":"code","source":["ecoli_blattman_copy"],"metadata":{"id":"9qtqu5sUG3ve"},"execution_count":null,"outputs":[]},{"cell_type":"code","source":["#sc.tl.rank_genes_groups(ecoli_blattman_copy, 'express', method='wilcoxon')\n","#sc.pl.rank_genes_groups(ecoli_blattman_copy)"],"metadata":{"id":"K-Zv1gcZH-n4"},"execution_count":null,"outputs":[]},{"cell_type":"code","source":["#markers_2 = 'oppABCDF','pdhR-aceEF-lpd'\n","#sce.pl.wishbone_marker_trajectory(ecoli_blattman_copy, markers_2)"],"metadata":{"id":"z58PiXJ4AFdj"},"execution_count":null,"outputs":[]},{"cell_type":"markdown","source":["# Differential Gene Expression"],"metadata":{"id":"djj7x0EHjK08"}},{"cell_type":"markdown","source":["Output to file then coordinate with the \"Wishbone Scanorama\""],"metadata":{"id":"-seCsfLqTRnB"}},{"cell_type":"code","source":["sc.tl.leiden(ecoli_blattman_copy, resolution = 0.3)"],"metadata":{"id":"NuyI_BUtvDU8"},"execution_count":null,"outputs":[]},{"cell_type":"code","source":["ecoli_blattman_copy.uns['leiden']"],"metadata":{"id":"Y_ECIRgFy5Jt","executionInfo":{"status":"ok","timestamp":1650231768081,"user_tz":240,"elapsed":8,"user":{"displayName":"Jacob Reis Rosenfeld","userId":"16476830039630475050"}},"colab":{"base_uri":"https://localhost:8080/"},"outputId":"69470f8e-59b4-45a4-e04b-e8fc5ed81eda"},"execution_count":null,"outputs":[{"output_type":"execute_result","data":{"text/plain":["{'params': {'n_iterations': -1, 'random_state': 0, 'resolution': 0.3}}"]},"metadata":{},"execution_count":21}]},{"cell_type":"code","source":["!pip install scprep\n","import scprep\n","\n","scprep.stats.differential_expression_by_cluster(ecoli_blattman_copy.X, ecoli_blattman_copy.obs['leiden'],gene_names = ecoli_blattman_copy.var_names)"],"metadata":{"id":"NNTaTckFjN9Y","executionInfo":{"status":"ok","timestamp":1650231772339,"user_tz":240,"elapsed":4264,"user":{"displayName":"Jacob Reis Rosenfeld","userId":"16476830039630475050"}},"colab":{"base_uri":"https://localhost:8080/"},"outputId":"be974072-1264-4657-cba6-25c63d1d8eec"},"execution_count":null,"outputs":[{"output_type":"stream","name":"stdout","text":["Requirement already satisfied: scprep in /usr/local/lib/python3.7/dist-packages (1.1.0)\n","Requirement already satisfied: pandas>=0.25 in /usr/local/lib/python3.7/dist-packages (from scprep) (1.3.5)\n","Requirement already satisfied: decorator>=4.3.0 in /usr/local/lib/python3.7/dist-packages (from scprep) (4.4.2)\n","Requirement already satisfied: scikit-learn>=0.19.1 in /usr/local/lib/python3.7/dist-packages (from scprep) (1.0.2)\n","Requirement already satisfied: packaging in /usr/local/lib/python3.7/dist-packages (from scprep) (21.3)\n","Requirement already satisfied: numpy>=1.12.0 in /usr/local/lib/python3.7/dist-packages (from scprep) (1.21.5)\n","Requirement already satisfied: scipy>=0.18.1 in /usr/local/lib/python3.7/dist-packages (from scprep) (1.4.1)\n","Requirement already satisfied: pytz>=2017.3 in /usr/local/lib/python3.7/dist-packages (from pandas>=0.25->scprep) (2018.9)\n","Requirement already satisfied: python-dateutil>=2.7.3 in /usr/local/lib/python3.7/dist-packages (from pandas>=0.25->scprep) (2.8.2)\n","Requirement already satisfied: six>=1.5 in /usr/local/lib/python3.7/dist-packages (from python-dateutil>=2.7.3->pandas>=0.25->scprep) (1.15.0)\n","Requirement already satisfied: joblib>=0.11 in /usr/local/lib/python3.7/dist-packages (from scikit-learn>=0.19.1->scprep) (1.1.0)\n","Requirement already satisfied: threadpoolctl>=2.0.0 in /usr/local/lib/python3.7/dist-packages (from scikit-learn>=0.19.1->scprep) (3.1.0)\n","Requirement already satisfied: pyparsing!=3.0.5,>=2.0.2 in /usr/local/lib/python3.7/dist-packages (from packaging->scprep) (3.0.8)\n"]},{"output_type":"execute_result","data":{"text/plain":["{'0':                                              difference  rank\n"," pdhR-aceEF-lpd                                -0.633377     0\n"," rplNXE-rpsNH-rplFR-rpsE-rpmD-rplO-secY-rpmJ   -0.598518     1\n"," cyoABCDE                                      -0.581553     2\n"," rpsJ-rplCDWB-rpsS-rplV-rpsC-rplP-rpmC-rpsQ    -0.569070     3\n"," rpsMKD-rpoA-rplQ                              -0.519751     4\n"," ...                                                 ...   ...\n"," mdtNOP                                        -0.000056  1978\n"," yibF                                          -0.000056  1979\n"," greB                                          -0.000055  1980\n"," yaiI                                           0.000039  1981\n"," serV-argVYZQ                                   0.000039  1982\n"," \n"," [1983 rows x 2 columns],\n"," '1':                                              difference  rank\n"," rplNXE-rpsNH-rplFR-rpsE-rpmD-rplO-secY-rpmJ    1.033799     0\n"," pdhR-aceEF-lpd                                 1.024407     1\n"," rpsJ-rplCDWB-rpsS-rplV-rpsC-rplP-rpmC-rpsQ     1.008112     2\n"," rplKAJL-rpoBC                                  0.908106     3\n"," rpsLG-fusA-tufA                                0.859319     4\n"," ...                                                 ...   ...\n"," dgkA                                           0.000057  1978\n"," yqhG                                           0.000057  1979\n"," yjfKLMC                                       -0.000033  1980\n"," lysU                                           0.000030  1981\n"," acrR                                          -0.000001  1982\n"," \n"," [1983 rows x 2 columns],\n"," '2':                      difference  rank\n"," tig                    0.925687     0\n"," gnd                    0.809081     1\n"," rplM-rpsI              0.769781     2\n"," pyrG-eno               0.669297     3\n"," rplU-rpmA-yhbE-obgE    0.594217     4\n"," ...                         ...   ...\n"," ybdG                   0.000275  1978\n"," ubiK                  -0.000201  1979\n"," argG                  -0.000200  1980\n"," idnDOTR               -0.000104  1981\n"," cysPUWAM               0.000033  1982\n"," \n"," [1983 rows x 2 columns],\n"," '3':                              difference  rank\n"," tolB-pal-cpoB                  1.317393     0\n"," cyoABCDE                       1.199948     1\n"," aroKB-damX-dam-rpe-gph-trpS    1.170537     2\n"," ompX                           1.156079     3\n"," rpsF-priB-rpsR-rplI            0.716797     4\n"," ...                                 ...   ...\n"," mdlAB                          0.000386  1978\n"," yegH                          -0.000246  1979\n"," fimAICDFGH                     0.000215  1980\n"," glrR-glnB                      0.000190  1981\n"," yfbS                          -0.000130  1982\n"," \n"," [1983 rows x 2 columns],\n"," '4':                                              difference  rank\n"," aspA-dcuA                                      2.875427     0\n"," rfbBDACX                                       2.721127     1\n"," ssrA                                           2.326610     2\n"," rplNXE-rpsNH-rplFR-rpsE-rpmD-rplO-secY-rpmJ   -0.409509     3\n"," rpsLG-fusA-tufA                               -0.391372     4\n"," ...                                                 ...   ...\n"," amn                                            0.000360  1978\n"," yeaGH                                         -0.000353  1979\n"," trpLEDCBA                                      0.000179  1980\n"," yegQ                                           0.000141  1981\n"," fadH                                          -0.000063  1982\n"," \n"," [1983 rows x 2 columns],\n"," '5':                                              difference  rank\n"," rRNA                                           2.382061     0\n"," rplNXE-rpsNH-rplFR-rpsE-rpmD-rplO-secY-rpmJ   -0.977418     1\n"," rpsJ-rplCDWB-rpsS-rplV-rpsC-rplP-rpmC-rpsQ    -0.909671     2\n"," rplKAJL-rpoBC                                 -0.807003     3\n"," rpsLG-fusA-tufA                               -0.768918     4\n"," ...                                                 ...   ...\n"," yidB                                          -0.003938  1978\n"," lysZ                                          -0.003929  1979\n"," ybhD                                          -0.003886  1980\n"," ygaY_1Y_2                                     -0.003824  1981\n"," pmrD                                          -0.003815  1982\n"," \n"," [1983 rows x 2 columns],\n"," '6':                                              difference  rank\n"," rRNA                                           2.375267     0\n"," rplNXE-rpsNH-rplFR-rpsE-rpmD-rplO-secY-rpmJ   -0.975006     1\n"," rpsJ-rplCDWB-rpsS-rplV-rpsC-rplP-rpmC-rpsQ    -0.907426     2\n"," rplKAJL-rpoBC                                 -0.805011     3\n"," rpsLG-fusA-tufA                               -0.767020     4\n"," ...                                                 ...   ...\n"," yidB                                          -0.003928  1978\n"," lysZ                                          -0.003919  1979\n"," ybhD                                          -0.003876  1980\n"," ygaY_1Y_2                                     -0.003814  1981\n"," pmrD                                          -0.003805  1982\n"," \n"," [1983 rows x 2 columns]}"]},"metadata":{},"execution_count":22}]},{"cell_type":"code","source":["sc.tl.rank_genes_groups(ecoli_blattman_copy, 'leiden', method = 't-test', key_added = 't-test')\n","sc.pl.rank_genes_groups(ecoli_blattman_copy, n_genes=10, sharey=False, key = \"t-test\")"],"metadata":{"id":"HWthoiZYzL7t","executionInfo":{"status":"ok","timestamp":1650231774245,"user_tz":240,"elapsed":1912,"user":{"displayName":"Jacob Reis Rosenfeld","userId":"16476830039630475050"}},"colab":{"base_uri":"https://localhost:8080/"},"outputId":"56900bbf-f273-4add-b017-644d14b6dd2a"},"execution_count":null,"outputs":[{"output_type":"stream","name":"stderr","text":["/usr/local/lib/python3.7/dist-packages/statsmodels/tools/_testing.py:19: FutureWarning: pandas.util.testing is deprecated. Use the functions in the public API at pandas.testing instead.\n","  import pandas.util.testing as tm\n","/usr/local/lib/python3.7/dist-packages/scanpy/tools/_rank_genes_groups.py:421: RuntimeWarning: invalid value encountered in log2\n","  foldchanges[global_indices]\n"]}]},{"cell_type":"code","source":["sc.tl.rank_genes_groups(ecoli_blattman_copy, 'leiden', method='t-test_overestim_var', key_added = \"t-test_ov\")\n","sc.pl.rank_genes_groups(ecoli_blattman_copy, n_genes=10, sharey=False, key = \"t-test_ov\")"],"metadata":{"id":"-OFfb6MnzXs_","executionInfo":{"status":"ok","timestamp":1650231775748,"user_tz":240,"elapsed":1507,"user":{"displayName":"Jacob Reis Rosenfeld","userId":"16476830039630475050"}},"colab":{"base_uri":"https://localhost:8080/"},"outputId":"14a5a2a6-7709-49fb-c492-2583ac134d29"},"execution_count":null,"outputs":[{"output_type":"stream","name":"stderr","text":["/usr/local/lib/python3.7/dist-packages/scanpy/tools/_rank_genes_groups.py:421: RuntimeWarning: invalid value encountered in log2\n","  foldchanges[global_indices]\n"]}]},{"cell_type":"code","source":["sc.tl.rank_genes_groups(ecoli_blattman_copy, 'leiden', method='wilcoxon', key_added = \"wilcoxon\")\n","sc.pl.rank_genes_groups(ecoli_blattman_copy, n_genes=10, sharey=False, key=\"wilcoxon\")"],"metadata":{"id":"z_HjxnTrzj_0","executionInfo":{"status":"ok","timestamp":1650231780669,"user_tz":240,"elapsed":4925,"user":{"displayName":"Jacob Reis Rosenfeld","userId":"16476830039630475050"}},"colab":{"base_uri":"https://localhost:8080/"},"outputId":"2f357fd9-bfda-4586-f9d1-021e84537db4"},"execution_count":null,"outputs":[{"output_type":"stream","name":"stderr","text":["/usr/local/lib/python3.7/dist-packages/scanpy/tools/_rank_genes_groups.py:421: RuntimeWarning: invalid value encountered in log2\n","  foldchanges[global_indices]\n"]}]},{"cell_type":"code","source":["sc.tl.rank_genes_groups(ecoli_blattman_copy, 'leiden', method='logreg',key_added = \"logreg\")\n","sc.pl.rank_genes_groups(ecoli_blattman_copy, n_genes=10, sharey=False, key = \"logreg\")"],"metadata":{"id":"2TEAksGHzszD","executionInfo":{"status":"ok","timestamp":1650231829550,"user_tz":240,"elapsed":48892,"user":{"displayName":"Jacob Reis Rosenfeld","userId":"16476830039630475050"}},"colab":{"base_uri":"https://localhost:8080/"},"outputId":"7dbc1925-4245-49e8-d08d-513e2abbbecb"},"execution_count":null,"outputs":[{"output_type":"stream","name":"stderr","text":["/usr/local/lib/python3.7/dist-packages/sklearn/linear_model/_logistic.py:818: ConvergenceWarning: lbfgs failed to converge (status=1):\n","STOP: TOTAL NO. of ITERATIONS REACHED LIMIT.\n","\n","Increase the number of iterations (max_iter) or scale the data as shown in:\n","    https://scikit-learn.org/stable/modules/preprocessing.html\n","Please also refer to the documentation for alternative solver options:\n","    https://scikit-learn.org/stable/modules/linear_model.html#logistic-regression\n","  extra_warning_msg=_LOGISTIC_SOLVER_CONVERGENCE_MSG,\n"]}]},{"cell_type":"code","source":["sc.tl.dendrogram(ecoli_blattman_copy, groupby = 'leiden')\n","sc.pl.rank_genes_groups_heatmap(ecoli_blattman_copy, n_genes=5, key=\"wilcoxon\", groupby=\"leiden\", show_gene_labels=True)"],"metadata":{"id":"sGKja7cdz43V"},"execution_count":null,"outputs":[]},{"cell_type":"code","source":["sc.pl.rank_genes_groups_dotplot(ecoli_blattman_copy, n_genes=5, key=\"wilcoxon\", groupby=\"leiden\")"],"metadata":{"id":"cmDxFVtJ0DuK"},"execution_count":null,"outputs":[]},{"cell_type":"code","source":["sc.pl.rank_genes_groups_stacked_violin(ecoli_blattman_copy, n_genes=5, key=\"wilcoxon\", groupby=\"leiden\")"],"metadata":{"id":"EPWUpgss0IcA"},"execution_count":null,"outputs":[]},{"cell_type":"code","source":["sc.pl.rank_genes_groups_matrixplot(ecoli_blattman_copy, n_genes=5, key=\"wilcoxon\", groupby=\"leiden\")"],"metadata":{"id":"yRgaQMny0Orh"},"execution_count":null,"outputs":[]},{"cell_type":"code","source":["ecoli_blattman_copy.write(filename = '/Colab Notebooks/Blattman Data Post TI')"],"metadata":{"id":"sJ86RRE7ctnA"},"execution_count":null,"outputs":[]}]}