1. download data
## 2022-05-02 17:07:31 URL:https://cf.10xgenomics.com/samples/cell-arc/1.0.0/pbmc_granulocyte_sorted_10k/pbmc_granulocyte_sorted_10k_atac_fragments.tsv.gz [2051027831/2051027831] -> "pbmc_granulocyte_sorted_10k_atac_fragments.tsv.gz" [1]
## 2022-05-02 17:07:32 URL:https://cf.10xgenomics.com/samples/cell-arc/1.0.0/pbmc_granulocyte_sorted_10k/pbmc_granulocyte_sorted_10k_atac_fragments.tsv.gz.tbi [1027204/1027204] -> "pbmc_granulocyte_sorted_10k_atac_fragments.tsv.gz.tbi" [1]
## 2022-05-02 17:07:33 URL:https://uc2e99488f9d608bc8cd77316676.dl.dropboxusercontent.com/cd/0/inline/BkiCO7CY1DavkuGDqXpdRi5IDk_bfz4QbyIorFGDgpRVz_sFTbOQqDqBAL75Ai4wLIYdz8IXIhsQ-wKGhSR7tkxmulUrXpTX2_n9exX93GHIZ8Ewd0ez3IV63O9-llNlVSAojpBLkkg08mBc0j76BZQEvY0BeCKuQ3CRbTvd4xogGA/file [400178/400178] -> "PBMC-Multiom_annotation.tsv" [1]
## 2022-05-02 17:08:08 URL:https://cf.10xgenomics.com/samples/cell-arc/1.0.0/pbmc_granulocyte_sorted_10k/pbmc_granulocyte_sorted_10k_filtered_feature_bc_matrix.h5 [162282142/162282142] -> "pbmc_granulocyte_sorted_10k_filtered_feature_bc_matrix.h5" [1]
2. library
## Setting default genome to Hg38.
## Input threads is equal to or greater than ncores minus 1 (7)
## Setting cores to ncores minus 2. Set force = TRUE to set above this number!
## Setting default number of Parallel threads to 6.
3. Create ArchR proj
inputFiles <- "./pbmc_granulocyte_sorted_10k_atac_fragments.tsv.gz"
ArrowFiles <- createArrowFiles(inputFiles = inputFiles,
sampleNames = "pbmc",
minTSS = 0, ##4
minFrags = 0, ## 1000
addTileMat = T,
addGeneScoreMat = F)
## Using GeneAnnotation set by addArchRGenome(Hg38)!
## Using GeneAnnotation set by addArchRGenome(Hg38)!
## ArchR logging to : ArchRLogs/ArchR-createArrows-71002bd84768-Date-2022-05-02_Time-17-08-19.log
## If there is an issue, please report to github with logFile!
## Cleaning Temporary Files
## 2022-05-02 17:08:19 : Batch Execution w/ safelapply!, 0 mins elapsed.
## (pbmc : 1 of 1) Determining Arrow Method to use!
## 2022-05-02 17:08:19 : (pbmc : 1 of 1) Reading In Fragments from inputFiles (readMethod = tabix), 0 mins elapsed.
## 2022-05-02 17:08:19 : (pbmc : 1 of 1) Tabix Bed To Temporary File, 0 mins elapsed.
## 2022-05-02 17:18:21 : (pbmc : 1 of 1) Successful creation of Temporary File, 10.047 mins elapsed.
## 2022-05-02 17:18:21 : (pbmc : 1 of 1) Creating ArrowFile From Temporary File, 10.047 mins elapsed.
## 2022-05-02 17:20:16 : (pbmc : 1 of 1) Successful creation of Arrow File, 11.956 mins elapsed.
## 2022-05-02 17:21:47 : (pbmc : 1 of 1) CellStats : Number of Cells Pass Filter = 639351 , 13.473 mins elapsed.
## 2022-05-02 17:21:47 : (pbmc : 1 of 1) CellStats : Median Frags = 5 , 13.473 mins elapsed.
## 2022-05-02 17:21:47 : (pbmc : 1 of 1) CellStats : Median TSS Enrichment = 0.099 , 13.473 mins elapsed.
## WARNING: Error found with Cairo installation. Continuing without rasterization.
## 2022-05-02 17:22:01 : (pbmc : 1 of 1) Adding Additional Feature Counts!, 13.705 mins elapsed.
## 2022-05-02 17:23:21 : (pbmc : 1 of 1) Removing Fragments from Filtered Cells, 15.042 mins elapsed.
## 2022-05-02 17:23:21 : (pbmc : 1 of 1) Creating Filtered Arrow File, 15.042 mins elapsed.
## 2022-05-02 17:24:10 : (pbmc : 1 of 1) Finished Constructing Filtered Arrow File!, 15.86 mins elapsed.
## 2022-05-02 17:24:12 : (pbmc : 1 of 1) Adding TileMatrix!, 15.898 mins elapsed.
## 2022-05-02 17:26:12 : (pbmc : 1 of 1) Finished Creating Arrow File, 17.899 mins elapsed.
## ArchR logging successful to : ArchRLogs/ArchR-createArrows-71002bd84768-Date-2022-05-02_Time-17-08-19.log
ArrowFiles <- "pbmc.arrow"
proj <- ArchRProject(ArrowFiles = ArrowFiles, outputDirectory = "PBMC",
copyArrows = T, showLogo=F)
## Using GeneAnnotation set by addArchRGenome(Hg38)!
## Using GeneAnnotation set by addArchRGenome(Hg38)!
## Validating Arrows...
## Getting SampleNames...
## 1
## Copying ArrowFiles to Ouptut Directory! If you want to save disk space set copyArrows = FALSE
## 1
## Getting Cell Metadata...
## 1
## Merging Cell Metadata...
## Initializing ArchRProject...
meta <- read.csv("PBMC-Multiom_annotation.tsv", sep="\t")
rownames(meta) <- paste0("pbmc#", (meta$barcode))
bc <- intersect(rownames(proj@cellColData), rownames(meta))
meta <- meta[bc, ]
proj <- proj[bc, ]
proj <- setCellCol(proj, meta=meta)
gene.coords <- genes(EnsDb.Hsapiens.v86, filter = ~ gene_biotype == "protein_coding")
ucsc.levels <- stringr::str_replace(string=paste("chr",seqlevels(gene.coords),sep=""), pattern="chrMT", replacement="chrM")
seqlevels(gene.coords) <- ucsc.levels
genebody.coords <- keepStandardChromosomes(gene.coords, pruning.mode = 'coarse')
mtxs <- Read10X_h5("pbmc_granulocyte_sorted_10k_filtered_feature_bc_matrix.h5")
## Genome matrix has multiple modalities, returning a list of matrices for this genome
## ArchR logging to : ArchRLogs/ArchR-addGeneExpressionMatrix-710041238866-Date-2022-05-02_Time-17-26-31.log
## If there is an issue, please report to github with logFile!
## Overlap w/ scATAC = 1
## 2022-05-02 17:26:32 :
## Overlap Per Sample w/ scATAC : pbmc=11763
## 2022-05-02 17:26:32 :
## 2022-05-02 17:26:33 : Batch Execution w/ safelapply!, 0 mins elapsed.
## 2022-05-02 17:26:34 : Adding pbmc to GeneExpressionMatrix for Chr (1 of 23)!, 0.016 mins elapsed.
## 2022-05-02 17:26:35 : Adding pbmc to GeneExpressionMatrix for Chr (2 of 23)!, 0.039 mins elapsed.
## 2022-05-02 17:26:37 : Adding pbmc to GeneExpressionMatrix for Chr (3 of 23)!, 0.058 mins elapsed.
## 2022-05-02 17:26:38 : Adding pbmc to GeneExpressionMatrix for Chr (4 of 23)!, 0.086 mins elapsed.
## 2022-05-02 17:26:39 : Adding pbmc to GeneExpressionMatrix for Chr (5 of 23)!, 0.106 mins elapsed.
## 2022-05-02 17:26:41 : Adding pbmc to GeneExpressionMatrix for Chr (6 of 23)!, 0.126 mins elapsed.
## 2022-05-02 17:26:42 : Adding pbmc to GeneExpressionMatrix for Chr (7 of 23)!, 0.152 mins elapsed.
## 2022-05-02 17:26:43 : Adding pbmc to GeneExpressionMatrix for Chr (8 of 23)!, 0.171 mins elapsed.
## 2022-05-02 17:26:44 : Adding pbmc to GeneExpressionMatrix for Chr (9 of 23)!, 0.19 mins elapsed.
## 2022-05-02 17:26:46 : Adding pbmc to GeneExpressionMatrix for Chr (10 of 23)!, 0.217 mins elapsed.
## 2022-05-02 17:26:47 : Adding pbmc to GeneExpressionMatrix for Chr (11 of 23)!, 0.237 mins elapsed.
## 2022-05-02 17:26:48 : Adding pbmc to GeneExpressionMatrix for Chr (12 of 23)!, 0.256 mins elapsed.
## 2022-05-02 17:26:50 : Adding pbmc to GeneExpressionMatrix for Chr (13 of 23)!, 0.284 mins elapsed.
## 2022-05-02 17:26:51 : Adding pbmc to GeneExpressionMatrix for Chr (14 of 23)!, 0.303 mins elapsed.
## 2022-05-02 17:26:52 : Adding pbmc to GeneExpressionMatrix for Chr (15 of 23)!, 0.321 mins elapsed.
## 2022-05-02 17:26:54 : Adding pbmc to GeneExpressionMatrix for Chr (16 of 23)!, 0.348 mins elapsed.
## 2022-05-02 17:26:55 : Adding pbmc to GeneExpressionMatrix for Chr (17 of 23)!, 0.367 mins elapsed.
## 2022-05-02 17:26:56 : Adding pbmc to GeneExpressionMatrix for Chr (18 of 23)!, 0.388 mins elapsed.
## 2022-05-02 17:26:58 : Adding pbmc to GeneExpressionMatrix for Chr (19 of 23)!, 0.414 mins elapsed.
## 2022-05-02 17:26:59 : Adding pbmc to GeneExpressionMatrix for Chr (20 of 23)!, 0.434 mins elapsed.
## 2022-05-02 17:27:00 : Adding pbmc to GeneExpressionMatrix for Chr (21 of 23)!, 0.453 mins elapsed.
## 2022-05-02 17:27:02 : Adding pbmc to GeneExpressionMatrix for Chr (22 of 23)!, 0.479 mins elapsed.
## 2022-05-02 17:27:03 : Adding pbmc to GeneExpressionMatrix for Chr (23 of 23)!, 0.498 mins elapsed.
## ArchR logging successful to : ArchRLogs/ArchR-addGeneExpressionMatrix-710041238866-Date-2022-05-02_Time-17-26-31.log
4. RNA & ATAC dimension reductions
proj <- addIterativeLSI(ArchRProj = proj,
useMatrix = "TileMatrix",
name = "IterativeLSI",
iterations = 2,
varFeatures = 25000,
dimsToUse = 1:30)
## Checking Inputs...
## ArchR logging to : ArchRLogs/ArchR-addIterativeLSI-71004d2ffa79-Date-2022-05-02_Time-17-27-05.log
## If there is an issue, please report to github with logFile!
## 2022-05-02 17:27:06 : Computing Total Across All Features, 0 mins elapsed.
## 2022-05-02 17:27:06 : Computing Top Features, 0.008 mins elapsed.
## ###########
## 2022-05-02 17:27:07 : Running LSI (1 of 2) on Top Features, 0.025 mins elapsed.
## ###########
## 2022-05-02 17:27:07 : Sampling Cells (N = 10000) for Estimated LSI, 0.026 mins elapsed.
## 2022-05-02 17:27:07 : Creating Sampled Partial Matrix, 0.026 mins elapsed.
## 2022-05-02 17:27:32 : Computing Estimated LSI (projectAll = FALSE), 0.433 mins elapsed.
## 2022-05-02 17:28:22 : Identifying Clusters, 1.269 mins elapsed.
## 2022-05-02 17:28:32 : Identified 6 Clusters, 1.433 mins elapsed.
## 2022-05-02 17:28:32 : Saving LSI Iteration, 1.433 mins elapsed.
## WARNING: Error found with Cairo installation. Continuing without rasterization.
## WARNING: Error found with Cairo installation. Continuing without rasterization.
## 2022-05-02 17:28:43 : Creating Cluster Matrix on the total Group Features, 1.618 mins elapsed.
## 2022-05-02 17:29:27 : Computing Variable Features, 2.355 mins elapsed.
## ###########
## 2022-05-02 17:29:27 : Running LSI (2 of 2) on Variable Features, 2.356 mins elapsed.
## ###########
## 2022-05-02 17:29:27 : Creating Partial Matrix, 2.356 mins elapsed.
## 2022-05-02 17:29:49 : Computing LSI, 2.725 mins elapsed.
## 2022-05-02 17:30:04 : Finished Running IterativeLSI, 2.982 mins elapsed.
object <- CreateSeuratObject(counts=RNA, assay="RNA")
object <- NormalizeData(object, normalization.method = "LogNormalize", scale.factor = 10000, verbose=F)
object <- FindVariableFeatures(object, nfeatures=3000, verbose=F)
object <- ScaleData(object, verbose=F)
object <- RunPCA(object, npcs=50, reduction.name="RNA_PCA", verbose=F)
proj <- setDimRed(proj, mtx=Embeddings(object[["RNA_PCA"]]), type="reducedDims", reduction.name="RNA_PCA")
saveArchRProject(proj)
## Saving ArchRProject...
## Loading ArchRProject...
## Successfully loaded ArchRProject!
##
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## /__/ \__\ | _| `._____| \______||__| |__| | _| `._____|
##
## class: ArchRProject
## outputDirectory: /Users/zhijianli/Github/MOJITOO/vignettes/PBMC
## samples(1): pbmc
## sampleColData names(1): ArrowFiles
## cellColData names(20): Sample nMultiFrags ... Gex_MitoRatio
## Gex_RiboRatio
## numberOfCells(1): 11763
## medianTSS(1): 13.659
## medianFrags(1): 13486
5. RUN MOJITOO
## Successfully loaded ArchRProject!
proj <- mojitoo(
object=proj,
reduction.list = list("RNA_PCA", "IterativeLSI"),
dims.list = list(1:50, 1:30),
is.reduction.center=T,
is.reduction.scale=T,
reduction.name='MOJITOO'
)
## processing RNA_PCA
## adding IterativeLSI
## 1 round cc 29
proj <- addUMAP(
ArchRProj = proj,
reducedDims = "MOJITOO",
name = "MOJITOO_UMAP",
nNeighbors = 30,
minDist = 0.5,
metric = "cosine"
)
## 17:30:17 UMAP embedding parameters a = 0.583 b = 1.334
## 17:30:17 Read 11763 rows and found 29 numeric columns
## 17:30:17 Using Annoy for neighbor search, n_neighbors = 30
## 17:30:17 Building Annoy index with metric = cosine, n_trees = 50
## 0% 10 20 30 40 50 60 70 80 90 100%
## [----|----|----|----|----|----|----|----|----|----|
## **************************************************|
## 17:30:17 Writing NN index file to temp file /var/folders/75/g5lhn91d62bgnv_gd4x0fc240000gn/T//RtmpCOg6B1/file71003cb22889
## 17:30:17 Searching Annoy index using 4 threads, search_k = 3000
## 17:30:18 Annoy recall = 100%
## 17:30:19 Commencing smooth kNN distance calibration using 4 threads
## 17:30:20 Initializing from normalized Laplacian + noise
## 17:30:20 Commencing optimization for 200 epochs, with 462816 positive edges
## 17:30:24 Optimization finished
## 17:30:24 Creating temp model dir /var/folders/75/g5lhn91d62bgnv_gd4x0fc240000gn/T//RtmpCOg6B1/dir71007b690dd8
## 17:30:24 Creating dir /var/folders/75/g5lhn91d62bgnv_gd4x0fc240000gn/T//RtmpCOg6B1/dir71007b690dd8
## 17:30:25 Changing to /var/folders/75/g5lhn91d62bgnv_gd4x0fc240000gn/T//RtmpCOg6B1/dir71007b690dd8
## 17:30:25 Creating /Users/zhijianli/Github/MOJITOO/vignettes/PBMC/Embeddings/Save-Uwot-UMAP-Params-MOJITOO-710054835980-Date-2022-05-02_Time-17-30-24.tar
6. UMAP of True labels
p <- plotEmbedding(ArchRProj = proj, colorBy = "cellColData", name = "annotation", embedding = "MOJITOO_UMAP")
## ArchR logging to : ArchRLogs/ArchR-plotEmbedding-71001dd4a68a-Date-2022-05-02_Time-17-30-25.log
## If there is an issue, please report to github with logFile!
## Getting UMAP Embedding
## ColorBy = cellColData
## Plotting Embedding
## 1 WARNING: Error found with Cairo installation. Continuing without rasterization.
##
## ArchR logging successful to : ArchRLogs/ArchR-plotEmbedding-71001dd4a68a-Date-2022-05-02_Time-17-30-25.log
7. sessionInfo
## R version 4.2.0 (2022-04-22)
## Platform: aarch64-apple-darwin21.3.0 (64-bit)
## Running under: macOS Monterey 12.3.1
##
## Matrix products: default
## BLAS: /opt/homebrew/Cellar/openblas/0.3.20/lib/libopenblasp-r0.3.20.dylib
## LAPACK: /opt/homebrew/Cellar/r/4.2.0/lib/R/lib/libRlapack.dylib
##
## locale:
## [1] C/UTF-8/C/C/C/C
##
## attached base packages:
## [1] grid parallel stats4 stats graphics grDevices utils
## [8] datasets methods base
##
## other attached packages:
## [1] uwot_0.1.11 nabor_0.5.0
## [3] gridExtra_2.3 Rsamtools_2.12.0
## [5] BSgenome.Hsapiens.UCSC.hg38_1.4.4 BSgenome_1.64.0
## [7] rtracklayer_1.56.0 Biostrings_2.64.0
## [9] XVector_0.36.0 EnsDb.Hsapiens.v86_2.99.0
## [11] ensembldb_2.20.0 AnnotationFilter_1.20.0
## [13] GenomicFeatures_1.48.0 AnnotationDbi_1.58.0
## [15] stringr_1.4.0 ggsci_2.9
## [17] sp_1.4-7 SeuratObject_4.1.0
## [19] Seurat_4.1.1 ArchR_1.0.1
## [21] magrittr_2.0.3 rhdf5_2.40.0
## [23] Matrix_1.4-1 data.table_1.14.2
## [25] SummarizedExperiment_1.26.0 Biobase_2.56.0
## [27] GenomicRanges_1.48.0 GenomeInfoDb_1.32.1
## [29] IRanges_2.30.0 S4Vectors_0.34.0
## [31] BiocGenerics_0.42.0 MatrixGenerics_1.8.0
## [33] matrixStats_0.62.0 ggplot2_3.3.5
## [35] MOJITOO_1.0
##
## loaded via a namespace (and not attached):
## [1] rappdirs_0.3.3 scattermore_0.8 ragg_1.2.2
## [4] tidyr_1.2.0 bit64_4.0.5 knitr_1.39
## [7] irlba_2.3.5 DelayedArray_0.22.0 rpart_4.1.16
## [10] KEGGREST_1.36.0 RCurl_1.98-1.6 doParallel_1.0.17
## [13] generics_0.1.2 cowplot_1.1.1 RSQLite_2.2.13
## [16] RANN_2.6.1 future_1.25.0 bit_4.0.4
## [19] spatstat.data_2.2-0 xml2_1.3.3 httpuv_1.6.5
## [22] assertthat_0.2.1 xfun_0.30 hms_1.1.1
## [25] jquerylib_0.1.4 evaluate_0.15 promises_1.2.0.1
## [28] fansi_1.0.3 restfulr_0.0.13 progress_1.2.2
## [31] dbplyr_2.1.1 igraph_1.3.1 DBI_1.1.2
## [34] htmlwidgets_1.5.4 spatstat.geom_2.4-0 purrr_0.3.4
## [37] ellipsis_0.3.2 RSpectra_0.16-1 ks_1.13.5
## [40] dplyr_1.0.9 backports_1.4.1 biomaRt_2.52.0
## [43] deldir_1.0-6 vctrs_0.4.1 here_1.0.1
## [46] ROCR_1.0-11 abind_1.4-5 withr_2.5.0
## [49] cachem_1.0.6 Gviz_1.40.0 progressr_0.10.0
## [52] checkmate_2.1.0 sctransform_0.3.3 GenomicAlignments_1.32.0
## [55] prettyunits_1.1.1 mclust_5.4.9 goftest_1.2-3
## [58] cluster_2.1.3 lazyeval_0.2.2 crayon_1.5.1
## [61] hdf5r_1.3.5 labeling_0.4.2 pkgconfig_2.0.3
## [64] nlme_3.1-157 ProtGenerics_1.28.0 nnet_7.3-17
## [67] rlang_1.0.2 globals_0.14.0 lifecycle_1.0.1
## [70] miniUI_0.1.1.1 filelock_1.0.2 BiocFileCache_2.4.0
## [73] dichromat_2.0-0.1 rprojroot_2.0.3 polyclip_1.10-0
## [76] lmtest_0.9-40 Rhdf5lib_1.18.0 zoo_1.8-10
## [79] base64enc_0.1-3 ggridges_0.5.3 GlobalOptions_0.1.2
## [82] png_0.1-7 viridisLite_0.4.0 rjson_0.2.21
## [85] bitops_1.0-7 KernSmooth_2.23-20 rhdf5filters_1.8.0
## [88] blob_1.2.3 shape_1.4.6 parallelly_1.31.1
## [91] spatstat.random_2.2-0 jpeg_0.1-9 scales_1.2.0
## [94] memoise_2.0.1 plyr_1.8.7 ica_1.0-2
## [97] zlibbioc_1.42.0 hdrcde_3.4 compiler_4.2.0
## [100] BiocIO_1.6.0 RColorBrewer_1.1-3 clue_0.3-60
## [103] fitdistrplus_1.1-8 cli_3.3.0 listenv_0.8.0
## [106] patchwork_1.1.1 pbapply_1.5-0 htmlTable_2.4.0
## [109] Formula_1.2-4 MASS_7.3-57 mgcv_1.8-40
## [112] tidyselect_1.1.2 stringi_1.7.6 textshaping_0.3.6
## [115] highr_0.9 yaml_2.3.5 latticeExtra_0.6-29
## [118] ggrepel_0.9.1 sass_0.4.1 VariantAnnotation_1.42.0
## [121] tools_4.2.0 future.apply_1.9.0 circlize_0.4.14
## [124] rstudioapi_0.13 foreach_1.5.2 foreign_0.8-82
## [127] farver_2.1.0 Rtsne_0.16 digest_0.6.29
## [130] rgeos_0.5-9 pracma_2.3.8 shiny_1.7.1
## [133] Rcpp_1.0.8.3 later_1.3.0 RcppAnnoy_0.0.19
## [136] fda_6.0.3 httr_1.4.2 biovizBase_1.44.0
## [139] ComplexHeatmap_2.12.0 colorspace_2.0-3 rainbow_3.6
## [142] XML_3.99-0.9 fs_1.5.2 tensor_1.5
## [145] reticulate_1.24 splines_4.2.0 spatstat.utils_2.3-0
## [148] pkgdown_2.0.3 plotly_4.10.0 systemfonts_1.0.4
## [151] xtable_1.8-4 fds_1.8 jsonlite_1.8.0
## [154] corpcor_1.6.10 R6_2.5.1 Hmisc_4.7-0
## [157] ramify_0.3.3 pillar_1.7.0 htmltools_0.5.2
## [160] mime_0.12 glue_1.6.2 fastmap_1.1.0
## [163] BiocParallel_1.30.0 deSolve_1.32 codetools_0.2-18
## [166] pcaPP_2.0-1 mvtnorm_1.1-3 utf8_1.2.2
## [169] lattice_0.20-45 bslib_0.3.1 spatstat.sparse_2.1-1
## [172] tibble_3.1.6 curl_4.3.2 leiden_0.3.10
## [175] gtools_3.9.2 survival_3.3-1 rmarkdown_2.14
## [178] desc_1.4.1 munsell_0.5.0 GetoptLong_1.0.5
## [181] GenomeInfoDbData_1.2.8 iterators_1.0.14 reshape2_1.4.4
## [184] gtable_0.3.0 spatstat.core_2.4-2