CytoTRACE2
CytoTRACE2是 Newman Lab 开发的用于预测scRNA-seq数据中细胞干性和发育潜能的计算方法。
CytoTRACE2本质是一种深度学习框架，该模型在涵盖 28 种组织类型和整个发育范围的人类和小鼠 scRNA-seq 数据集的训练和验证（总共31个数据）。利用该模型，CytoTRACE2可以将细胞分类为终末分化（differentiated: 0）和全能细胞（totipotent: 1）.
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本文简单介绍CytoTRACE2 R包的基本用法，具体原理可参考他们目前的预印本 https://doi.org/10.1101/2024.03.19.585637
安装
devtools::install_github("digitalcytometry/cytotrace2", subdir = "cytotrace2_r") #installing
数据准备
CytoTRACE2需要的输入数据包括单细胞表达数据，这里利用一个已发表的胰腺的10X scRNA-seq作为示例 （Bastidas-Ponce et al., 2019）
这里下载CytoTRACE2提供的示例数据集，包括了：
-
表达数据（
expression_data）：小鼠胰腺上皮的2280细胞的10X scRNA-seq数据。该数据已经进行归一化处理 -
注释数据（
annotation）：表型注释
# download the .rds file (this will download the file to your working directory)
download.file("https://drive.google.com/uc?export=download&id=1ivi9TBlmzVTDGzNWQrXXeyL68Wug989K", "Pancreas_10x_downsampled.rds")
运行CytoTRACE2
运行 cytotrace2() 进行细胞干性推断，函数运行时执行以下操作：
-
预处理数据：在每个细胞中对表达数据进行降序排序，使用排序后的秩（rank）进行预测
-
预测细胞发育状态：读入模型预训练参数进行预测
# full model parameter_dict <- readRDS(system.file("extdata", "parameter_dict_17.rds", package = "CytoTRACE2")) # else parameter_dict <- readRDS(system.file("extdata", "parameter_dict_5_best.rds", package = "CytoTRACE2")) -
后处理预测结果：利用diffusion方法平滑预测值，并利用kNN和binning的方法重新正则化预测值
library(CytoTRACE2) #loading
## Loading required package: data.table
## Loading required package: doParallel
## Loading required package: foreach
## Loading required package: iterators
## Loading required package: parallel
## Loading required package: dplyr
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## Attaching package: 'dplyr'
## The following objects are masked from 'package:data.table':
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## between, first, last
## The following objects are masked from 'package:stats':
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## The following objects are masked from 'package:base':
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## Loading required package: ggplot2
## Loading required package: HiClimR
## Loading required package: magrittr
## Loading required package: Matrix
## Loading required package: plyr
## ------------------------------------------------------------------------------
## You have loaded plyr after dplyr - this is likely to cause problems.
## If you need functions from both plyr and dplyr, please load plyr first, then dplyr:
## library(plyr); library(dplyr)
## ------------------------------------------------------------------------------
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## Attaching package: 'plyr'
## The following objects are masked from 'package:dplyr':
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## Loading required package: RANN
## Loading required package: Rfast
## Loading required package: Rcpp
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## Attaching package: 'Rfast'
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## Loading required package: Seurat
## Loading required package: SeuratObject
## Loading required package: sp
## 'SeuratObject' was built under R 4.4.0 but the current version is
## 4.4.1; it is recomended that you reinstall 'SeuratObject' as the ABI
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## Attaching package: 'SeuratObject'
## The following objects are masked from 'package:base':
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## intersect, t
## Loading required package: stringr
## Warning: replacing previous import 'data.table::first' by 'dplyr::first' when
## loading 'CytoTRACE2'
## Warning: replacing previous import 'data.table::last' by 'dplyr::last' when
## loading 'CytoTRACE2'
## Warning: replacing previous import 'data.table::between' by 'dplyr::between'
## when loading 'CytoTRACE2'
# load rds
scdata <- readRDS("Pancreas_10x_downsampled.rds")
# extract expression data
expression_data <- scdata$expression_data
dim(expression_data)
## [1] 17326 2280
# running CytoTRACE 2 main function - cytotrace2 - with default parameters
cytotrace2_result <- cytotrace2(expression_data,
species = 'mouse',
is_seurat = F)
## cytotrace2: Started loading data
## Dataset contains 17326 genes and 2280 cells.
## The passed subsample size is greater than the number of cells in dataset.
## Now setting subsample size to 2280
## cytotrace2: Running on 1 subsample(s) approximately of length 2280
## cytotrace2: Started running on subsample(s). This will take a few minutes.
## cytotrace2: Started preprocessing.
## 13191 input genes mapped to model genes.
## cytotrace2: Started prediction.
## This section will run using 5 / 11 core(s).
## cytotrace2: Started postprocessing.
## cytotrace2: Running with fast mode (subsamples are processed in parallel)
## This section will run on 2 sub-sample(s) of approximately 1140 cells each using 2 / 11 core(s).
## cytotrace2: Finished
# extract annotation data
annotation <- scdata$annotation
head(annotation)
## phenotype1 phenotype2 potency
## CACCAGGCAGGTGCCT-1-0 Alpha cell Alpha Differentiated
## CCCAGTTAGGGAACGG-1-2 Alpha cell Alpha Differentiated
## CGGACTGCATCTACGA-1-2 Epsilon cell Epsilon Differentiated
## TACCTTAGTTCCAACA-1-2 Alpha cell Alpha Differentiated
## TACTTACCAGACAGGT-1-2 Alpha cell Alpha Differentiated
## ATGAGGGGTGGTACAG-1-3 Beta cell Beta Differentiated
## absolute_granular_ordering relative_ordering
## CACCAGGCAGGTGCCT-1-0 23 6
## CCCAGTTAGGGAACGG-1-2 23 6
## CGGACTGCATCTACGA-1-2 23 6
## TACCTTAGTTCCAACA-1-2 23 6
## TACTTACCAGACAGGT-1-2 23 6
## ATGAGGGGTGGTACAG-1-3 23 6
# generate prediction and phenotype association plots with plotData function
plots <- plotData(cytotrace2_result = cytotrace2_result,
annotation = annotation,
expression_data = expression_data
)
## Preparing input for visualization.
## Creating plots.
## Done. You can access any plot directly from the returned list by '$' operator (i.e. plots$CytoTRACE2_Potency_UMAP).
在plotData 这一步会运行seurat流程包括Normalization, FindVariableFeatures, PCA, UMAP.
# pseudocode
seurat <- CreateSeuratObject(counts = as.matrix(expression_data), project = "nn", min.cells = 3, min.features = 200)
# Normalization
seurat <- NormalizeData(seurat, normalization.method = "LogNormalize", scale.factor = 10000) ##defaults
# Find variable features
seurat <- FindVariableFeatures(seurat, selection.method = "vst", nfeatures = 2000)
# Scale data
all.genes <- rownames(seurat)
seurat <- ScaleData(seurat, features = all.genes)
# PCA
seurat <- RunPCA(seurat, features = VariableFeatures(object = seurat))
# UMAP
seurat <- RunUMAP(seurat, dims = 1:pc_dims) # default to 30
然后，将cytoTRACE2预测的细胞发育结果进行可视化，包括：
-
potency_score_umap：UMAP可视化cytoTRACE2预测的细胞潜能打分（
CytoTRACE2_Score） -
potency_category_umap：UMAP可视化cytoTRACE2预测的细胞干性分类（
CytoTRACE2_Potency） -
rel_order_umap：UMAP可视化cytoTRACE2预测的细胞潜能打分的相对排序值0-1（
CytoTRACE2_Relative） -
phenotype_umap：UMAP可视化表型分组，如果有的话。
-
potencyBoxplot_byPheno：每个表型分组的
CytoTRACE2_Score分布
table(annotation$phenotype1)
##
## Alpha cell Beta cell
## 276 258
## Delta cell Endocrine precursor cell
## 20 509
## Endocrine progenitor Epsilon cell
## 499 46
## Immature endocrine cell Multipotent pancreatic progenitor
## 427 245
table(cytotrace2_result$CytoTRACE2_Potency)
##
## Differentiated Unipotent Oligopotent Multipotent Pluripotent
## 952 598 501 229 0
## Totipotent
## 0
plots$CytoTRACE2_UMAP
unnamed-chunk-5-1.png
Potency score distribution by phenotype
plots$CytoTRACE2_Boxplot_byPheno
unnamed-chunk-6-1.png
Potency category
plots$CytoTRACE2_Potency_UMAP
unnamed-chunk-7-1.png
Relative order
plots$CytoTRACE2_Relative_UMAP
unnamed-chunk-8-1.png
Phenotype
plots$Phenotype_UMAP
unnamed-chunk-9-1.png
Seurat object input
library(Seurat)
seu <- CreateSeuratObject(expression_data, meta.data = annotation)
## Warning: Data is of class data.frame. Coercing to dgCMatrix.
seu
## An object of class Seurat
## 17326 features across 2280 samples within 1 assay
## Active assay: RNA (17326 features, 0 variable features)
## 1 layer present: counts
run cytotrace2
cytotrace2_result <- cytotrace2(seu, is_seurat = TRUE, slot_type = "counts", species = 'mouse')
## cytotrace2: Started loading data
## Dataset contains 17326 genes and 2280 cells.
## The passed subsample size is greater than the number of cells in dataset.
## Now setting subsample size to 2280
## cytotrace2: Running on 1 subsample(s) approximately of length 2280
## cytotrace2: Started running on subsample(s). This will take a few minutes.
## cytotrace2: Started preprocessing.
## 13191 input genes mapped to model genes.
## cytotrace2: Started prediction.
## This section will run using 5 / 11 core(s).
## cytotrace2: Started postprocessing.
## cytotrace2: Running with fast mode (subsamples are processed in parallel)
## This section will run on 2 sub-sample(s) of approximately 1140 cells each using 2 / 11 core(s).
## cytotrace2: Finished
# plotting
plots <- plotData(cytotrace2_result = cytotrace2_result,
annotation = annotation,
is_seurat = TRUE)
## Preparing input for visualization.
## Creating plots.
## Done. You can access any plot directly from the returned list by '$' operator (i.e. plots$CytoTRACE2_Potency_UMAP).
可视化
plots$CytoTRACE2_Potency_UMAP
unnamed-chunk-12-1.png
也可以用seurat流程跑完后的对象进行UMAP可视化
# Normalize the data
cytotrace2_result <- NormalizeData(cytotrace2_result)
## Normalizing layer: counts
# Find variable features
cytotrace2_result <- FindVariableFeatures(cytotrace2_result, selection.method = "vst", nfeatures = 2000)
## Finding variable features for layer counts
all.genes <- rownames(cytotrace2_result)
# Scale the data
cytotrace2_result <- ScaleData(cytotrace2_result, features=all.genes)
## Centering and scaling data matrix
# Perform PCA
cytotrace2_result <- RunPCA(cytotrace2_result, npcs = 30)
## PC_ 1
## Positive: Cpe, Pcsk1n, Pax6, Chga, Fam183b, Hmgn3, Chgb, Scg3, Map1b, Cryba2
## Isl1, Slc38a5, Neurod1, Prnp, Gnao1, Scgn, Gnas, Gch1, Aplp1, Fev
## Dbpht2, Pcsk2, Vwa5b2, Pyy, Pam, Pim2, Cnih2, Mafb, Abcc8, Tspan7
## Negative: H19, Wfdc2, Tead2, Sparc, Smco4, Phgdh, Rbp1, Rpl36a, Cd24a, Mgst1
## Adamts1, 1700011H14Rik, Gsta3, Stmn1, Ldha, Rpl39, Hmga2, Spp1, Ptn, Rpl12
## Galk1, Wfdc15b, Cdkn1c, Sox9, Serpinh1, Sox4, Habp2, Hes1, Vim, Rps12
## PC_ 2
## Positive: Btbd17, Ppp1r14a, Gadd45a, Neurod2, Igfbpl1, Sult2b1, Mfng, Neurog3, Pax4, Tubb3
## Btg2, Mfap4, Smarcd2, Tmsb4x, Plk3, Cbfa2t3, Cck, Notum, Actg1, Hes6
## Sulf2, Lynx1, Gfra3, Epb42, Lingo1, Gdpd1, Foxa3, Cdc14b, Hpca, Rcor2
## Negative: Atp1b1, Rbp4, Tagln2, Tuba1b, Akr1c19, Id2, Gcsh, Fxyd6, Rnase4, Bex4
## 1700011H14Rik, Tmem27, Siva1, Phgdh, Tubb2a, Meis2, Cmtm8, Mgst3, Ociad2, Crip1
## Adamts1, Parm1, Fkbp2, Fabp3, Id3, Eif4ebp1, Spc25, Sdc4, Cpn1, Cyr61
## PC_ 3
## Positive: Rpl13a, Rpl14, Rps14, mt-Atp6, Eef1a1, Rpl10, Rpl41, Ftl1, mt-Co3, Rps16
## Rps18, Rps27a, Rps3a1, Rpl26, mt-Cytb, Rps6, mt-Nd1, Rpl13, Rpl11, Rpl6
## Rpl9, mt-Co1, Rps5, Rps7, Ppia, Rps2, Rpl17, Rplp0, Rps9, Rpl32
## Negative: Neurog3, Anxa2, Sparc, Col9a3, Col9a1, Fxyd2, Amotl2, Mpc1, Aurka, Tpx2
## Cotl1, Ckb, Cenpf, Gpx2, Mia, Spp1, Cd82, Cdk2ap1, Cdc20, Lurap1l
## Pbk, Gsta3, Racgap1, Hmmr, Cxcl12, Aurkb, Ccnb1, Aldh1b1, Tpm1, Tpm4
## PC_ 4
## Positive: Cldn4, Runx1t1, Fev, Irf2bpl, Cacna2d1, Hnrnpa1, Pdk3, Slc35d3, Grb10, Gm43861
## Akr1c19, Jun, 1110012L19Rik, BC023829, Glud1, Vdr, Tm4sf4, Emb, St18, Celf3
## Pgf, Ids, Pcyt1b, Cited2, Maff, Elavl4, Sulf1, Neurod1, Serpini1, Ddc
## Negative: Ppp1r1a, Iapp, Pdia6, Scg2, Calr, Ttr, Higd1a, G6pc2, Tmed3, Tmsb15l
## Papss2, Sdf2l1, Hspa5, Clu, Ins1, Tmem27, Ly6e, Cd82, Slc30a8, Dap
## Igfbp7, Ins2, Ociad2, Gcg, Ttc28, Qsox1, Bcl2, Gapdh, Irx1, Npy
## PC_ 5
## Positive: Cxcl12, Pdzk1ip1, Anxa2, Grin3a, Dcdc2a, Krt18, 8430408G22Rik, Pamr1, Anxa5, Bicc1
## Capg, Muc1, Krt8, Krt7, S100a10, Cldn3, Cystm1, Spp1, Gsta3, Trim47
## Smoc2, Ppp1r1b, Col9a3, Vim, Ndrg2, Tsc22d1, Cat, Cldn4, Txnip, Nfib
## Negative: Vtn, 2810417H13Rik, Hist1h2ap, Chst2, Tuba1a, Top2a, P2rx1, Aurkb, Rrm2, Fbxo5
## Ccna2, Cpn1, Spc25, H2afz, Cenpf, Birc5, Ccnb1, Pbk, Gcat, Smc2
## Ube2c, Incenp, Mki67, Car14, Rad51ap1, Gc, Cdk1, Plk1, Prc1, Smc4
# Find neighbors
cytotrace2_result <- FindNeighbors(cytotrace2_result, dims = 1:20)
## Computing nearest neighbor graph
## Computing SNN
# Find clusters
cytotrace2_result <- FindClusters(cytotrace2_result, resolution = 0.1)
## Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
##
## Number of nodes: 2280
## Number of edges: 73179
##
## Running Louvain algorithm...
## Maximum modularity in 10 random starts: 0.9505
## Number of communities: 4
## Elapsed time: 0 seconds
# Run UMAP
cytotrace2_result <- RunUMAP(cytotrace2_result, dims = 1:20)
## 14:12:35 UMAP embedding parameters a = 0.9922 b = 1.112
## 14:12:35 Read 2280 rows and found 20 numeric columns
## 14:12:35 Using Annoy for neighbor search, n_neighbors = 30
## 14:12:35 Building Annoy index with metric = cosine, n_trees = 50
## 0% 10 20 30 40 50 60 70 80 90 100%
## [----|----|----|----|----|----|----|----|----|----|
## **************************************************|
## 14:12:35 Writing NN index file to temp file /var/folders/yk/896pglhn555drshbgn27f71h0000gn/T//RtmpD9l2F4/file95af46be469e
## 14:12:35 Searching Annoy index using 1 thread, search_k = 3000
## 14:12:35 Annoy recall = 100%
## 14:12:35 Commencing smooth kNN distance calibration using 1 thread with target n_neighbors = 30
## 14:12:35 Initializing from normalized Laplacian + noise (using RSpectra)
## 14:12:35 Commencing optimization for 500 epochs, with 87006 positive edges
## 14:12:37 Optimization finished
DimPlot(cytotrace2_result, group.by = 'CytoTRACE2_Potency')
unnamed-chunk-13-1.png
或者看score的分布
FeaturePlot(cytotrace2_result, features = 'CytoTRACE2_Score')
unnamed-chunk-14-1.png
Ref:
https://github.com/digitalcytometry/cytotrace2/tree/main
https://www.biorxiv.org/content/10.1101/2024.03.19.585637v1.full
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