单细胞分析工具--Palantir轨迹分析

Paper：https://www.nature.com/articles/s41587-019-0068-4

github：https://github.com/dpeerlab/Palantir

tutorial：https://nbviewer.org/github/dpeerlab/Palantir/blob/master/notebooks/Palantir_sample_notebook.ipynb

简介：Palantir是一个2019年在nature biotechnology提出的用于单细胞数据轨迹推断的Python工具包。根据官方教程，简单学习用法如下。

0、安装包与准备数据

1）安装python包

根据前期探索，不建议直接使用pip安装(存在bug)，而是下载github的最新源码后安装。

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unzip Palantir-master
cd Palantir-master
pip install .

2）示例数据

需要准备**.h5ad**的单细胞数据（count表达矩阵）格式，官方提供示例数据可直接下载。

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wget https://dp-lab-data-public.s3.amazonaws.com/palantir/marrow_sample_scseq_counts.h5ad

Seurat对象转为h5ad格式，参考笔记：***

1、加载包与环境

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import palantir
import scanpy as sc
import numpy as np
import pandas as pd
import os

# Plotting 
import matplotlib
import matplotlib.pyplot as plt
import seaborn as sns
import warnings

# Inline plotting
%matplotlib inline

sns.set_style('ticks')
matplotlib.rcParams['figure.figsize'] = [4, 4]
matplotlib.rcParams['figure.dpi'] = 100
matplotlib.rcParams['image.cmap'] = 'Spectral_r'
warnings.filterwarnings(action="ignore", module="matplotlib", message="findfont")

# Reset random seed
np.random.seed(5)

2、导入数据与预处理

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## (1) 导入数据
ad = sc.read('marrow_sample_scseq_counts.h5ad')
ad
# AnnData object with n_obs × n_vars = 4142 × 16106
# 4142个细胞，16106个基因

## (2) 标准化
sc.pp.normalize_per_cell(ad)
palantir.preprocess.log_transform(ad)

## (3) 高变基因
sc.pp.highly_variable_genes(ad, n_top_genes=1500, flavor='cell_ranger')

降维：PCA → UMAP

可以使用scanpy包的相关函数，也可以导入Seurat的降维结果，以保证可视化的一致性。

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## (1) scanpy
#PCA降维
sc.pp.pca(ad)
#UMAP降维
sc.pp.neighbors(ad)
sc.tl.umap(ad)

## (2) Seurat
seurat_pca = pd.read_csv("marrow_sample_scseq_pca.csv", index_col=0).values
ad.obsm['X_pca'] = seurat_pca
seurat_umap = pd.read_csv("marrow_sample_scseq_umap.csv", index_col=0).values
ad.obsm['X_umap'] = seurat_umap

ad
# AnnData object with n_obs × n_vars = 4142 × 16106
#     obs: 'n_counts'
#     var: 'highly_variable', 'means', 'dispersions', 'dispersions_norm'
#     uns: 'hvg', 'pca', 'neighbors', 'umap'
#     obsm: 'X_pca', 'X_umap'
#     varm: 'PCs'
#     obsp: 'distances', 'connectivities'


pca_projections = pd.DataFrame(ad.obsm['X_pca'], index=ad.obs_names)
umap = pd.DataFrame(ad.obsm['X_umap'], index=ad.obs_names)
# umap可视化
sc.pl.embedding(ad, basis='umap')

3、轨迹推断分析

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## (1)Run diffusion maps
dm_res = palantir.utils.run_diffusion_maps(pca_projections, n_components=5)

ms_data = palantir.utils.determine_multiscale_space(dm_res)
ad.layers['MAGIC_imputed_data'] = palantir.utils.run_magic_imputation(ad, dm_res)

#基因表达量可视化
sc.pl.embedding(ad, basis='umap', layer='MAGIC_imputed_data',
               color=['CD34', 'MPO', 'GATA1', 'IRF8'])

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## (2)轨迹推断
# 定义发育起点(required)
start_cell = 'Run5_164698952452459'

# 定义可能的发育终点(optional)
terminal_states = pd.Series(['DC', 'Mono', 'Ery'], 
                           index=['Run5_131097901611291', 
                                  'Run5_134936662236454', 
                                  'Run4_200562869397916'])
# 轨迹推断
pr_res = palantir.core.run_palantir(ms_data, start_cell, num_waypoints=500, terminal_states=terminal_states.index)
pr_res.branch_probs.columns = terminal_states[pr_res.branch_probs.columns]

# 基本可视化
palantir.plot.plot_palantir_results(pr_res, umap)

4、结果可视化

指定细胞的不同轨迹状态可能性

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cells = ['Run5_164698952452459', 'Run5_170327461775790', 'Run4_121896095574750', ]
palantir.plot.plot_terminal_state_probs(pr_res, cells) 

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palantir.plot.highlight_cells_on_tsne(umap, cells)

指定基因在不同发育路线的变化趋势

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genes = ['CD34', 'MPO', 'GATA1', 'IRF8']
imp_df = pd.DataFrame(ad[:, genes].layers['MAGIC_imputed_data'],
                     index=ad.obs_names, columns=genes)
gene_trends = palantir.presults.compute_gene_trends( pr_res, imp_df.loc[:, genes])

## 线图可视化
palantir.plot.plot_gene_trends(gene_trends)

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## 热图可视化
palantir.plot.plot_gene_trend_heatmaps(gene_trends)

选取特定基因，根据表达模式聚类

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genes = ad.var_names[:1000]
imp_df = pd.DataFrame(ad[:, genes].layers['MAGIC_imputed_data'], 
                     index=ad.obs_names, columns=genes)
gene_trends = palantir.presults.compute_gene_trends(pr_res, 
                    imp_df.iloc[:, 0:1000], ['Ery'])
trends = gene_trends['Ery']['trends']
gene_clusters = palantir.presults.cluster_gene_trends(trends)

palantir.plot.plot_gene_trend_clusters(trends, gene_clusters)

最后如原文所述，由于自身算法限制，本工具可能不太适合应用于肿瘤细胞生长、迁移等存在dedifferetiate或者trans-differentiate to earlier transcriptional state的发育轨迹推断

0、安装包与准备数据#

1）安装python包#

2）示例数据#

1、加载包与环境#

2、导入数据与预处理#

3、轨迹推断分析#

4、结果可视化#