examples/seurat4_pbmc3k.ipynb

{
 "cells": [
  {
   "cell_type": "code",
   "execution_count": 6,
   "metadata": {},
   "outputs": [],
   "source": [
    "# https://github.com/mojaveazure/seurat-disk/issues/48\n",
    "\n",
    "# https://mojaveazure.github.io/seurat-disk/articles/convert-anndata.html"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "# Test demo case"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 1,
   "metadata": {},
   "outputs": [
    {
     "name": "stderr",
     "output_type": "stream",
     "text": [
      "Attaching SeuratObject\n",
      "\n",
      "Registered S3 method overwritten by 'cli':\n",
      "  method     from         \n",
      "  print.boxx spatstat.geom\n",
      "\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "── \u001b[1mInstalled datasets\u001b[22m ───────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────── SeuratData v0.2.1 ──\n",
      "\n",
      "\u001b[32m✔\u001b[39m \u001b[34mpbmc3k\u001b[39m 3.1.4                                                                                            \n",
      "\n",
      "\n",
      "──────────────────────────────────────────────────────────────────────────────────────────────────────── Key ───────────────────────────────────────────────────────────────────────────────────────────────────────\n",
      "\n",
      "\u001b[32m✔\u001b[39m Dataset loaded successfully\n",
      "\u001b[33m❯\u001b[39m Dataset built with a newer version of Seurat than installed\n",
      "\u001b[31m❓\u001b[39m Unknown version of Seurat installed\n",
      "\n",
      "\n",
      "Registered S3 method overwritten by 'SeuratDisk':\n",
      "  method            from  \n",
      "  as.sparse.H5Group Seurat\n",
      "\n"
     ]
    }
   ],
   "source": [
    "library(Seurat)\n",
    "library(SeuratData)\n",
    "library(SeuratDisk)"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 4,
   "metadata": {},
   "outputs": [
    {
     "name": "stderr",
     "output_type": "stream",
     "text": [
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n",
      "Warning message in if (is.na(desc)) {:\n",
      "“the condition has length > 1 and only the first element will be used”\n"
     ]
    },
    {
     "data": {
      "text/plain": [
       "An object of class Seurat \n",
       "13714 features across 2638 samples within 1 assay \n",
       "Active assay: RNA (13714 features, 2000 variable features)\n",
       " 2 dimensional reductions calculated: pca, umap"
      ]
     },
     "metadata": {},
     "output_type": "display_data"
    }
   ],
   "source": [
    "InstallData(\"pbmc3k\")\n",
    "data(\"pbmc3k.final\")\n",
    "pbmc3k.final"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 7,
   "metadata": {},
   "outputs": [
    {
     "name": "stderr",
     "output_type": "stream",
     "text": [
      "Creating h5Seurat file for version 3.1.5.9900\n",
      "\n",
      "Adding counts for RNA\n",
      "\n",
      "Adding data for RNA\n",
      "\n",
      "Adding scale.data for RNA\n",
      "\n",
      "Adding variable features for RNA\n",
      "\n",
      "Adding feature-level metadata for RNA\n",
      "\n",
      "Adding cell embeddings for pca\n",
      "\n",
      "Adding loadings for pca\n",
      "\n",
      "No projected loadings for pca\n",
      "\n",
      "Adding standard deviations for pca\n",
      "\n",
      "Adding JackStraw information for pca\n",
      "\n",
      "Adding cell embeddings for umap\n",
      "\n",
      "No loadings for umap\n",
      "\n",
      "No projected loadings for umap\n",
      "\n",
      "No standard deviations for umap\n",
      "\n",
      "No JackStraw data for umap\n",
      "\n",
      "Validating h5Seurat file\n",
      "\n",
      "Adding scale.data from RNA as X\n",
      "\n",
      "Transfering meta.features to var\n",
      "\n",
      "Adding data from RNA as raw\n",
      "\n",
      "Transfering meta.features to raw/var\n",
      "\n",
      "Transfering meta.data to obs\n",
      "\n",
      "Adding dimensional reduction information for pca\n",
      "\n",
      "Adding feature loadings for pca\n",
      "\n",
      "Adding dimensional reduction information for umap\n",
      "\n",
      "Adding RNA_snn as neighbors\n",
      "\n"
     ]
    }
   ],
   "source": [
    "SaveH5Seurat(pbmc3k.final, filename = \"pbmc3k.h5Seurat\")\n",
    "Convert(\"pbmc3k.h5Seurat\", dest = \"h5ad\")"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "# Test on data from https://github.com/pinellolab/singlecellvr/blob/master/examples/seurat_10xpbmc.ipynb"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 2,
   "metadata": {},
   "outputs": [
    {
     "name": "stderr",
     "output_type": "stream",
     "text": [
      "\n",
      "Attaching package: ‘dplyr’\n",
      "\n",
      "\n",
      "The following objects are masked from ‘package:stats’:\n",
      "\n",
      "    filter, lag\n",
      "\n",
      "\n",
      "The following objects are masked from ‘package:base’:\n",
      "\n",
      "    intersect, setdiff, setequal, union\n",
      "\n",
      "\n",
      "Warning message:\n",
      "“Feature names cannot have underscores ('_'), replacing with dashes ('-')”\n"
     ]
    },
    {
     "data": {
      "text/plain": [
       "An object of class Seurat \n",
       "13714 features across 2700 samples within 1 assay \n",
       "Active assay: RNA (13714 features, 0 variable features)"
      ]
     },
     "metadata": {},
     "output_type": "display_data"
    }
   ],
   "source": [
    "library(dplyr)\n",
    "library(Seurat)\n",
    "library(patchwork)\n",
    "\n",
    "# Load the PBMC dataset\n",
    "pbmc.data <- Read10X(data.dir = \"/Users/qq06/Documents/singlecellvr/examples/filtered_gene_bc_matrices/hg19/\")\n",
    "# Initialize the Seurat object with the raw (non-normalized data).\n",
    "pbmc <- CreateSeuratObject(counts = pbmc.data, project = \"pbmc3k\", min.cells = 3, min.features = 200)\n",
    "pbmc"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 3,
   "metadata": {},
   "outputs": [],
   "source": [
    "pbmc[[\"percent.mt\"]] <- PercentageFeatureSet(pbmc, pattern = \"^MT-\")\n",
    "pbmc <- subset(pbmc, subset = nFeature_RNA > 200 & nFeature_RNA < 2500 & percent.mt < 5)"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 4,
   "metadata": {},
   "outputs": [],
   "source": [
    "pbmc <- NormalizeData(pbmc, normalization.method = \"LogNormalize\", scale.factor = 10000)"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 5,
   "metadata": {},
   "outputs": [],
   "source": [
    "pbmc <- FindVariableFeatures(pbmc, selection.method = \"vst\", nfeatures = 2000)\n"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 6,
   "metadata": {},
   "outputs": [
    {
     "name": "stderr",
     "output_type": "stream",
     "text": [
      "Centering and scaling data matrix\n",
      "\n"
     ]
    }
   ],
   "source": [
    "all.genes <- rownames(pbmc)\n",
    "pbmc <- ScaleData(pbmc, features = all.genes)"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 7,
   "metadata": {},
   "outputs": [
    {
     "name": "stderr",
     "output_type": "stream",
     "text": [
      "PC_ 1 \n",
      "Positive:  CST3, TYROBP, LST1, AIF1, FTL, FTH1, LYZ, FCN1, S100A9, TYMP \n",
      "\t   FCER1G, CFD, LGALS1, S100A8, CTSS, LGALS2, SERPINA1, IFITM3, SPI1, CFP \n",
      "\t   PSAP, IFI30, SAT1, COTL1, S100A11, NPC2, GRN, LGALS3, GSTP1, PYCARD \n",
      "Negative:  MALAT1, LTB, IL32, IL7R, CD2, B2M, ACAP1, CD27, STK17A, CTSW \n",
      "\t   CD247, GIMAP5, AQP3, CCL5, SELL, TRAF3IP3, GZMA, MAL, CST7, ITM2A \n",
      "\t   MYC, GIMAP7, HOPX, BEX2, LDLRAP1, GZMK, ETS1, ZAP70, TNFAIP8, RIC3 \n",
      "PC_ 2 \n",
      "Positive:  CD79A, MS4A1, TCL1A, HLA-DQA1, HLA-DQB1, HLA-DRA, LINC00926, CD79B, HLA-DRB1, CD74 \n",
      "\t   HLA-DMA, HLA-DPB1, HLA-DQA2, CD37, HLA-DRB5, HLA-DMB, HLA-DPA1, FCRLA, HVCN1, LTB \n",
      "\t   BLNK, P2RX5, IGLL5, IRF8, SWAP70, ARHGAP24, FCGR2B, SMIM14, PPP1R14A, C16orf74 \n",
      "Negative:  NKG7, PRF1, CST7, GZMB, GZMA, FGFBP2, CTSW, GNLY, B2M, SPON2 \n",
      "\t   CCL4, GZMH, FCGR3A, CCL5, CD247, XCL2, CLIC3, AKR1C3, SRGN, HOPX \n",
      "\t   TTC38, APMAP, CTSC, S100A4, IGFBP7, ANXA1, ID2, IL32, XCL1, RHOC \n",
      "PC_ 3 \n",
      "Positive:  HLA-DQA1, CD79A, CD79B, HLA-DQB1, HLA-DPB1, HLA-DPA1, CD74, MS4A1, HLA-DRB1, HLA-DRA \n",
      "\t   HLA-DRB5, HLA-DQA2, TCL1A, LINC00926, HLA-DMB, HLA-DMA, CD37, HVCN1, FCRLA, IRF8 \n",
      "\t   PLAC8, BLNK, MALAT1, SMIM14, PLD4, LAT2, IGLL5, P2RX5, SWAP70, FCGR2B \n",
      "Negative:  PPBP, PF4, SDPR, SPARC, GNG11, NRGN, GP9, RGS18, TUBB1, CLU \n",
      "\t   HIST1H2AC, AP001189.4, ITGA2B, CD9, TMEM40, PTCRA, CA2, ACRBP, MMD, TREML1 \n",
      "\t   NGFRAP1, F13A1, SEPT5, RUFY1, TSC22D1, MPP1, CMTM5, RP11-367G6.3, MYL9, GP1BA \n",
      "PC_ 4 \n",
      "Positive:  HLA-DQA1, CD79B, CD79A, MS4A1, HLA-DQB1, CD74, HLA-DPB1, HIST1H2AC, PF4, TCL1A \n",
      "\t   SDPR, HLA-DPA1, HLA-DRB1, HLA-DQA2, HLA-DRA, PPBP, LINC00926, GNG11, HLA-DRB5, SPARC \n",
      "\t   GP9, AP001189.4, CA2, PTCRA, CD9, NRGN, RGS18, GZMB, CLU, TUBB1 \n",
      "Negative:  VIM, IL7R, S100A6, IL32, S100A8, S100A4, GIMAP7, S100A10, S100A9, MAL \n",
      "\t   AQP3, CD2, CD14, FYB, LGALS2, GIMAP4, ANXA1, CD27, FCN1, RBP7 \n",
      "\t   LYZ, S100A11, GIMAP5, MS4A6A, S100A12, FOLR3, TRABD2A, AIF1, IL8, IFI6 \n",
      "PC_ 5 \n",
      "Positive:  GZMB, NKG7, S100A8, FGFBP2, GNLY, CCL4, CST7, PRF1, GZMA, SPON2 \n",
      "\t   GZMH, S100A9, LGALS2, CCL3, CTSW, XCL2, CD14, CLIC3, S100A12, CCL5 \n",
      "\t   RBP7, MS4A6A, GSTP1, FOLR3, IGFBP7, TYROBP, TTC38, AKR1C3, XCL1, HOPX \n",
      "Negative:  LTB, IL7R, CKB, VIM, MS4A7, AQP3, CYTIP, RP11-290F20.3, SIGLEC10, HMOX1 \n",
      "\t   PTGES3, LILRB2, MAL, CD27, HN1, CD2, GDI2, ANXA5, CORO1B, TUBA1B \n",
      "\t   FAM110A, ATP1A1, TRADD, PPA1, CCDC109B, ABRACL, CTD-2006K23.1, WARS, VMO1, FYB \n",
      "\n"
     ]
    }
   ],
   "source": [
    "pbmc <- RunPCA(pbmc, features = VariableFeatures(object = pbmc))"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 8,
   "metadata": {},
   "outputs": [
    {
     "name": "stderr",
     "output_type": "stream",
     "text": [
      "Computing nearest neighbor graph\n",
      "\n",
      "Computing SNN\n",
      "\n"
     ]
    },
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck\n",
      "\n",
      "Number of nodes: 2638\n",
      "Number of edges: 95965\n",
      "\n",
      "Running Louvain algorithm...\n",
      "Maximum modularity in 10 random starts: 0.8723\n",
      "Number of communities: 9\n",
      "Elapsed time: 0 seconds\n"
     ]
    }
   ],
   "source": [
    "pbmc <- FindNeighbors(pbmc, dims = 1:10)\n",
    "pbmc <- FindClusters(pbmc, resolution = 0.5)"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 11,
   "metadata": {},
   "outputs": [
    {
     "name": "stderr",
     "output_type": "stream",
     "text": [
      "10:10:49 UMAP embedding parameters a = 0.9922 b = 1.112\n",
      "\n",
      "10:10:49 Read 2638 rows and found 10 numeric columns\n",
      "\n",
      "10:10:49 Using Annoy for neighbor search, n_neighbors = 30\n",
      "\n",
      "10:10:49 Building Annoy index with metric = cosine, n_trees = 50\n",
      "\n",
      "0%   10   20   30   40   50   60   70   80   90   100%\n",
      "\n",
      "[----|----|----|----|----|----|----|----|----|----|\n",
      "\n",
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      "|\n",
      "\n",
      "10:10:49 Writing NN index file to temp file /var/folders/4m/79_qd_t11ldcnk_xm7bs96z80000gn/T//RtmpqWAdtj/file582b60eb6c75\n",
      "\n",
      "10:10:49 Searching Annoy index using 1 thread, search_k = 3000\n",
      "\n",
      "10:10:50 Annoy recall = 100%\n",
      "\n",
      "10:10:50 Commencing smooth kNN distance calibration using 1 thread\n",
      "\n",
      "10:10:50 Initializing from normalized Laplacian + noise\n",
      "\n",
      "10:10:50 Commencing optimization for 500 epochs, with 105124 positive edges\n",
      "\n",
      "10:10:54 Optimization finished\n",
      "\n"
     ]
    }
   ],
   "source": [
    "pbmc <- RunUMAP(pbmc, dims = 1:10, n.components = 3)"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 14,
   "metadata": {},
   "outputs": [
    {
     "name": "stderr",
     "output_type": "stream",
     "text": [
      "Creating h5Seurat file for version 3.1.5.9900\n",
      "\n",
      "Adding counts for RNA\n",
      "\n",
      "Adding data for RNA\n",
      "\n",
      "Adding scale.data for RNA\n",
      "\n",
      "Adding variable features for RNA\n",
      "\n",
      "Adding feature-level metadata for RNA\n",
      "\n",
      "Adding cell embeddings for pca\n",
      "\n",
      "Adding loadings for pca\n",
      "\n",
      "No projected loadings for pca\n",
      "\n",
      "Adding standard deviations for pca\n",
      "\n",
      "No JackStraw data for pca\n",
      "\n",
      "Adding cell embeddings for umap\n",
      "\n",
      "No loadings for umap\n",
      "\n",
      "No projected loadings for umap\n",
      "\n",
      "No standard deviations for umap\n",
      "\n",
      "No JackStraw data for umap\n",
      "\n",
      "Validating h5Seurat file\n",
      "\n",
      "Adding scale.data from RNA as X\n",
      "\n",
      "Transfering meta.features to var\n",
      "\n",
      "Adding data from RNA as raw\n",
      "\n",
      "Transfering meta.features to raw/var\n",
      "\n",
      "Transfering meta.data to obs\n",
      "\n",
      "Adding dimensional reduction information for pca\n",
      "\n",
      "Adding feature loadings for pca\n",
      "\n",
      "Adding dimensional reduction information for umap\n",
      "\n",
      "Adding RNA_snn as neighbors\n",
      "\n"
     ]
    }
   ],
   "source": [
    "SaveH5Seurat(pbmc, filename = \"demo.h5Seurat\")\n",
    "Convert(\"demo.h5Seurat\", dest = \"h5ad\")"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "metadata": {},
   "outputs": [],
   "source": []
  }
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