学习一篇NC的单细胞文章（一）：质控
学习一篇NC的单细胞文章（二）：标准化
学习一篇NC的单细胞文章（三）：复杂热图
学习一篇NC的单细胞文章（四）：Clustering
学习一篇NC的单细胞文章（五）：tSNE
在第五节，由于大部分细胞（868 个）都被归为上皮细胞群中（Fig2 c），这868个细胞可被分成5个cluster，接着对这5个cluster细胞进行探索。我们使用一组来自对乳腺肿块的非监督分析的基因表达特征对5个cluster进行了研究。这些基因表达特征通过比较三阴性乳腺癌（TNBC）的四个亚型(ERBB2 amplicon，Luminal Subtype 、Basal epithelial-cell enriched 和Luminal epithelial gene cluster containing ER)而建立。先看看这5个clusters的基底细胞来源的细胞群有多少。
## 读取数据
basal_PNAS_all <- read.table("data/genes_for_basal_vs_non_basal_tnbc_PNAS.txt", header = TRUE, sep = "\t")
#提取Basal.epithelial.cell.enriched.cluster的基因
basal_PNAS_long <- basal_PNAS_all$Basal.epithelial.cell.enriched.cluster
#合并剩下17个基因
basal_PNAS <- intersect(basal_PNAS_long, rownames(mat_ct))
> basal_PNAS
[1] "SOX9" "GALNT3" "CDH3" "LAMC2" "CX3CL1" "TRIM29" "KRT17" "KRT5" "CHI3L2"
[10] "SLPI" "NFIB" "MRAS" "TGFB2" "CAPN6" "DMD" "FABP7" "CXCL1"
#算出17个basal_PNAS基因在1112个细胞的表达平均值
basal_PNAS_avg_exprs <- apply(mat_ct[match(basal_PNAS, rownames(mat_ct)),], 2, mean)
#检查一下数据
all.equal(names(basal_PNAS_avg_exprs), colnames(mat_ct))
#提取868个上皮细胞群体的17个basal_PNAS基因表达平均值
basal_PNAS_avg_exprs <- basal_PNAS_avg_exprs[which(pd_ct$cell_types_cl_all == "epithelial")]
#检查一下数据
all.equal(colnames(HSMM_allepith_clustering), names(basal_PNAS_avg_exprs))
#把17个basal_PNAS基因表达平均值赋给HSMM_allepith_clustering，以便于后续分析
pData(HSMM_allepith_clustering)$basal_PNAS_avg_exprs <- basal_PNAS_avg_exprs
#画figS9b
plot_cell_clusters(HSMM_allepith_clustering, 1, 2, color = "basal_PNAS_avg_exprs", cell_size = 2) + facet_wrap(~patient) +
scale_color_continuous(low = "yellow", high = "blue")
figS9b
#画figS9a
plot_cell_clusters(HSMM_allepith_clustering, 1, 2, color = "basal_PNAS_avg_exprs", cell_size = 2) + facet_wrap(~Cluster) +
scale_color_continuous(low = "yellow", high = "blue")
figS9a
figS9a:大多数TNBC样本都有basal gene signature的表达。figS9b:在868个上皮细胞群中，cluster2的basal gene signature表达量最丰富。
接着，使用另外一个基因表达特征数据集TNBCtype4 signatures，这个signatures根据基因将细胞分为6个类：basal_like_1、basal_like_2、immunomodulatory、mesenchymal、mesenchymal_stem_like和luminal_ar。作者将基因表达特征中上调基因的平均表达值减去下调基因的平均表达值，将差值作为每个细胞在TNBCtype4 signatures （basal_like_1、basal_like_2、mesenchymal和luminal_ar）中的每个基因表达值，挑选最高基因表达值对应的signature，将其分配给对应的细胞。
#读取数据
lehman_long <- read.table("data/Lehman_signature.txt", sep = "\t", header = TRUE, stringsAsFactors = FALSE)
#这个for循环提取了lehman_long里面的四列gene、regulation、no_samples和signature，建立一个data.rrame
for (i in 0:5) {
gene <- "gene"
regulation <- "regulation"
no_samples <- "no_samples"
signature <- "signature"
if (i == 0) {
lehman <- lehman_long[, 1:4]
lehman <- lehman[-which(lehman$signature == ""),]
}
if (i > 0) {
gene <- paste("gene", i, sep = ".")
regulation <- paste("regulation", i, sep = ".")
no_samples <- paste("no_samples", i, sep = ".")
signature <- paste("signature", i, sep = ".")
mat_to_bind <- lehman_long[, c(gene, regulation, no_samples, signature)]
colnames(mat_to_bind) <- c("gene", "regulation", "no_samples", "signature")
if (length(which(is.na(mat_to_bind$no_samples))) > 0 )
mat_to_bind <- mat_to_bind[-which(mat_to_bind$signature == ""),]
lehman <- rbind(lehman, mat_to_bind)
}
}
#删掉一些mat_ct没有检测到的基因
lehman <- lehman[which(!is.na(match(lehman$gene, rownames(mat_ct)))),]
lehman_signatures <- unique(lehman$signature)
lehman_avg_exps <- apply(mat_ct, 2, function(x){
mns <- matrix(NA, nrow = length(lehman_signatures), ncol = 2)
rownames(mns) <- lehman_signatures
for (s in 1:length(lehman_signatures)) {
sign <- lehman_signatures[s] # current signature
lehman_here <- lehman %>%
dplyr::filter(signature == sign)
lehman_here_up <- lehman_here %>%
dplyr::filter(regulation == "UP")
lehman_here_down <- lehman_here %>%
dplyr::filter(regulation == "DOWN")
idx_genes_up <- match(lehman_here_up$gene, rownames(mat_ct))
idx_genes_down <- match(lehman_here_down$gene, rownames(mat_ct))
mns[s,] <- c(mean(x[idx_genes_up]), mean(x[idx_genes_down])) #算上调、下调的基因在样本中的平均表达值
}
return(mns)
})
#检查数据
all.equal(colnames(lehman_avg_exps), rownames(pd_ct))
#只看868个上皮细胞的情况
lehman_avg_exprs_epithelial <- lehman_avg_exps[,which(pd_ct$cell_types_cl_all == "epithelial")]
#提取lehman_avg_exps前面6行，对应的是up
lehman_avg_ups <- lehman_avg_exps[c(1:6), ]
rownames(lehman_avg_ups) <- lehman_signatures
all.equal(colnames(lehman_avg_ups), rownames(pd_ct))
lehman_avg_ups_epithelial <- lehman_avg_ups[,which(pd_ct$cell_types_cl_all == "epithelial")]
#提取lehman_avg_exps后面6行，对应的是down
lehman_avg_downs <- lehman_avg_exps[c(7:12),]
rownames(lehman_avg_downs) <- lehman_signatures
all.equal(colnames(lehman_avg_downs), rownames(pd_ct))
lehman_avg_downs_epithelial <- lehman_avg_downs[,which(pd_ct$cell_types_cl_all == "epithelial")]
#上调基因的平均表达值减去下调基因的平均表达值
lehman_avg_both <- lehman_avg_ups - lehman_avg_downs
all.equal(colnames(lehman_avg_both), rownames(pd_ct))
#挑选最高基因表达值对应的signature，将其分配给对应的细胞。
assignments_lehman_both <- apply(lehman_avg_both, 2, function(x){rownames(lehman_avg_both)[which.max(x)]})
assignments_lehman_both_epithelial <- assignments_lehman_both[which(pd_ct$cell_types_cl_all == "epithelial")]
#删除immunomodulatory和mesenchymal_stem_like signature
lehman_avg_both_epithelial_new <- lehman_avg_both_epithelial[-which(rownames(lehman_avg_both_epithelial) %in% c("immunomodulatory", "mesenchymal_stem_like")),]
assignments_lehman_both_epithelial_new <- apply(lehman_avg_both_epithelial_new, 2, function(x){rownames(lehman_avg_both_epithelial_new)[which.max(x)]})
接下来画图，同样地，需要对heatmap函数代码进行修改。
ha_lehman_epith_pat <- list()
for (i in 1:length(patients_now)) {
if (i == 1)
ha_lehman_epith_pat[[i]] <- HeatmapAnnotation(df=data.frame(cluster_all = clusterings_sep_allepith[[i]]),
col = list(cluster_all = c("1" = "#ee204d", "2" = "#17806d", "3" = "#b2ec5d", "4" = "#cda4de", "5" = "#1974d2")),
annotation_name_side = "left", annotation_name_gp = gpar(fontsize = 12),
annotation_legend_param = list(list(title_position = "topcenter", title = "cluster")),
show_annotation_name = FALSE,
gap = unit(c(2), "mm"),
show_legend = FALSE)
if (i > 1 && i != 5 )
ha_lehman_epith_pat[[i]] <- HeatmapAnnotation(df=data.frame(cluster_all = clusterings_sep_allepith[[i]]),
col = list(cluster_all = c("1" = "#ee204d", "2" = "#17806d", "3" = "#b2ec5d", "4" = "#cda4de", "5" = "#1974d2")),
annotation_name_side = "left", annotation_name_gp = gpar(fontsize = 12),
annotation_legend_param = list(list(title_position = "topcenter", title = "cluster")),
show_annotation_name = FALSE,
gap = unit(c(2), "mm"),
show_legend = FALSE)
if (i == 5)
ha_lehman_epith_pat[[i]] <- HeatmapAnnotation(df=data.frame(cluster_all = clusterings_sep_allepith[[i]]),
col = list(cluster_all = c("1" = "#ee204d", "2" = "#17806d", "3" = "#b2ec5d", "4" = "#cda4de", "5" = "#1974d2")),
annotation_name_side = "right", annotation_name_gp = gpar(fontsize = 12),
annotation_legend_param = list(list(title_position = "topcenter",title = "cluster")),
show_annotation_name = FALSE,
gap = unit(c(2), "mm"),
show_legend = TRUE)
}
#检查数据
all.equal(names(lehmans_epith_pat_both), patients_now)
#将basal signature添加进去，以便后续作图
lehmans_epith_pat_both_wbasal_new <- lehmans_epith_pat_both_new
for (i in 1:length(patients_now)) {
lehmans_epith_pat_both_wbasal_new[[i]] <- rbind(lehmans_epith_pat_both_new[[i]], pData(HSMM_allepith_clustering)$basal_PNAS_avg_exprs[which(HSMM_allepith_clustering$patient == patients_now[i])])
rownames(lehmans_epith_pat_both_wbasal_new[[i]])[5] <- "intrinsic_basal"
}
# 画图
ht_sep_lehmans_both_wbasal_new <-
Heatmap(lehmans_epith_pat_both_wbasal_new[[1]],
col = colorRamp2(c(-0.7, 0, 1), c("blue","white", "red")),
cluster_rows = FALSE,
show_column_names = FALSE,
column_title = patients_now[1],
column_title_gp = gpar(fontsize = 12),
top_annotation = ha_lehman_epith_pat[[1]],
name = patients_now[1],
show_row_names = FALSE,
heatmap_legend_param = list(title_gp = gpar(fontsize = 9), labels_gp = gpar(fontsize = 9))) +
Heatmap(lehmans_epith_pat_both_wbasal_new[[2]],
col = colorRamp2(c(-0.7, 0, 1), c("blue","white", "red")),
cluster_rows = FALSE,
show_column_names = FALSE,
column_title = patients_now[2],
column_title_gp = gpar(fontsize = 12),
top_annotation = ha_lehman_epith_pat[[2]],
name = patients_now[2],
show_row_names = FALSE,
heatmap_legend_param = list(title_gp = gpar(fontsize = 9), labels_gp = gpar(fontsize = 9))) +
Heatmap(lehmans_epith_pat_both_wbasal_new[[3]],
col = colorRamp2(c(-0.7, 0, 1), c("blue","white", "red")),
cluster_rows = FALSE,
show_column_names = FALSE,
column_title = patients_now[3],
column_title_gp = gpar(fontsize = 12),
top_annotation = ha_lehman_epith_pat[[3]],
name = patients_now[3],
show_row_names = FALSE,
heatmap_legend_param = list(title_gp = gpar(fontsize = 9), labels_gp = gpar(fontsize = 9))) +
Heatmap(lehmans_epith_pat_both_wbasal_new[[4]],
col = colorRamp2(c(-0.7, 0, 1), c("blue","white", "red")),
cluster_rows = FALSE,
show_column_names = FALSE,
column_title = patients_now[4],
column_title_gp = gpar(fontsize = 12),
top_annotation = ha_lehman_epith_pat[[4]],
name = patients_now[4],
show_row_names = FALSE,
heatmap_legend_param = list(title_gp = gpar(fontsize = 9), labels_gp = gpar(fontsize = 9))) +
Heatmap(lehmans_epith_pat_both_wbasal_new[[5]],
col = colorRamp2(c(-0.7, 0, 1), c("blue","white", "red")),
cluster_rows = FALSE,
show_column_names = FALSE,
column_title = patients_now[5],
column_title_gp = gpar(fontsize = 12),
top_annotation = ha_lehman_epith_pat[[5]],
name = patients_now[5],
show_row_names = FALSE,
heatmap_legend_param = list(title_gp = gpar(fontsize = 9), labels_gp = gpar(fontsize = 9))) +
Heatmap(lehmans_epith_pat_both_wbasal_new[[6]],
col = colorRamp2(c(-0.7, 0, 1), c("blue","white", "red")),
cluster_rows = FALSE,
row_names_side = "right",
column_title = patients_now[6],
column_title_gp = gpar(fontsize = 12),
top_annotation = ha_lehman_epith_pat[[6]],
name = patients_now[6],
show_column_names = FALSE,
top_annotation_height = unit(c(2), "cm"),
heatmap_legend_param = list(title_gp = gpar(fontsize = 9), labels_gp = gpar(fontsize = 9)))
#画fig3d
print(draw(ht_sep_lehmans_both_wbasal_new, annotation_legend_side = "right"))
fig3d
我们只需要把右边注释条PS一下，就可以达到跟文献的图片一模一样了。
#检查数据
all.equal(colnames(HSMM_allepith_clustering), names(assignments_lehman_both_epithelial_new))
pData(HSMM_allepith_clustering)$assignments_lehman_both_new <- assignments_lehman_both_epithelial_new
画fig3g
plot_cell_clusters(HSMM_allepith_clustering, 1, 2, color = "assignments_lehman_both_new", cell_size = 2) + facet_wrap(~patient)
fig3g
#画figS8
plot_cell_clusters(HSMM_allepith_clustering, 1, 2, color = "assignments_lehman_both_new", cell_size = 2) + facet_wrap(~Cluster)
figS8
cluster4也富集了 Basal-Like 1 signature，而cluster3高度富集了TNBCtype “Luminal Androgen Receptor” signature。为了更清楚的看到上皮细胞群的5个cluster对应的TNBCtype signatures的平均表达量，接着继续探索下去...
clust_avg_lehman_both_new <- matrix(NA, nrow = length(unique(HSMM_allepith_clustering$Cluster)), ncol = nrow(lehman_avg_both_epithelial_new))
#列名：cluster1,cluster2,cluster3,cluster4,cluster5
rownames(clust_avg_lehman_both_new) <- paste("clust", c(1:length(unique(HSMM_allepith_clustering$Cluster))), sep = "")
#行名：basal_like_1、basal_like_2、mesenchymal、luminal_ar"
colnames(clust_avg_lehman_both_new) <- rownames(lehman_avg_both_epithelial_new)
#算出每个cluster的signatures 平均值
for (c in 1:length(unique(HSMM_allepith_clustering$Cluster))) {
clust_avg_lehman_both_new[c,] <- apply(lehman_avg_both_epithelial_new[,which(HSMM_allepith_clustering$Cluster == c)], 1, mean)
}
clust_avg_lehman_both_new <- as.data.frame(clust_avg_lehman_both_new)
#增加一列cluster
clust_avg_lehman_both_new$Cluster <- rownames(clust_avg_lehman_both_new)
#拆分数据
clust_avg_lehman_melt_new <- melt(clust_avg_lehman_both_new, "Cluster")
#画fig3e
ggplot(clust_avg_lehman_melt_new, aes(Cluster, value, fill = factor(variable), color = factor(variable),
shape = factor(variable))) +
geom_point(size = 3, stroke = 1) +
scale_shape_discrete(solid = T) +
#guides(colour = guide_legend(override.aes = list(size=3))) +
ylab("average expression of signature in cluster") +
xlab("cluster") +
ylim(c(-0.35, 0.5))
fig3e
可以看到：cluster2和4强烈地表达Basal-Like 1 signature，而cluster3显著表达Basal-Like 2 signature和 luminal AR signature
接着，读取另外一个gene signatures，作者通过从上调基因的平均表达量中减去下调基因的平均表达量，计算出three normal breast signatures下每个细胞的表达量(Lim et al., 2009a)，然后将每个细胞分配给其具有最高表达量的signatures。这三个normal breast signatures 是：mature luminal (ML)，basal和luminal progenitor (LP)，在每个signatures中，都有对应的上调基因和下调基因。
## normal signatures
ml_signature_long <- read.table("data/ML_signature.txt", sep = "\t", header = TRUE)
if (length(which(ml_signature_long$Symbol == "")) > 0)
#将空白行去掉
ml_signature_long <- ml_signature_long[-which(ml_signature_long$Symbol == ""),]
#按照基因字母进行排序，如果基因字母有一样的，则按照Average.log.fold.change绝对值的负数进行从小到大排序
ml_signature_long <- ml_signature_long[order(ml_signature_long$Symbol, -abs(ml_signature_long$Average.log.fold.change) ), ]
#对基因取唯一值，去重复
ml_signature_long <- ml_signature_long[ !duplicated(ml_signature_long$Symbol), ]
#总共有818个基因
ml_signature <- ml_signature_long[which(!is.na(match(ml_signature_long$Symbol, rownames(mat_ct)))), ]
#上调基因有384个
ml_up <- ml_signature[which(ml_signature$Average.log.fold.change > 0), ]
#下调基因有197个
ml_down <- ml_signature[which(ml_signature$Average.log.fold.change < 0), ]
#匹配一下
idx_ml_up <- match(ml_up$Symbol, rownames(mat_ct))
idx_ml_down <- match(ml_down$Symbol, rownames(mat_ct))
#读取basal signature，处理过程跟上面的一样的。
basal_signature_long <- read.table("data/basal_signature.txt", sep = "\t", header = TRUE)
if (length(which(basal_signature_long$Symbol == "")) > 0)
basal_signature_long <- basal_signature_long[-which(basal_signature_long$Symbol == ""),]
basal_signature_long <- basal_signature_long[order(basal_signature_long$Symbol, -abs(basal_signature_long$Average.log.fold.change) ), ]
basal_signature_long <- basal_signature_long[ !duplicated(basal_signature_long$Symbol), ]
#总共有1335个基因
basal_signature <- basal_signature_long[which(!is.na(match(basal_signature_long$Symbol, rownames(mat_ct)))), ]
#上调基因有588个
basal_up <- basal_signature[which(basal_signature$Average.log.fold.change > 0), ]
#下调基因有757个
basal_down <- basal_signature[which(basal_signature$Average.log.fold.change < 0), ]
idx_basal_up <- match(basal_up$Symbol, rownames(mat_ct))
idx_basal_down <- match(basal_down$Symbol, rownames(mat_ct))
#读取LP signature,还是同样的操作
lp_signature_long <- read.table("data/Lp_signature.txt", sep = "\t", header = TRUE)
if (length(which(lp_signature_long$Symbol == "")) > 0)
lp_signature_long <- lp_signature_long[-which(lp_signature_long$Symbol == ""),]
lp_signature_long <- lp_signature_long[order(lp_signature_long$Symbol, -abs(lp_signature_long$Average.log.fold.change) ), ]
lp_signature_long <- lp_signature_long[ !duplicated(lp_signature_long$Symbol), ]
lp_signature <- lp_signature_long[which(!is.na(match(lp_signature_long$Symbol, rownames(mat_ct)))), ]
lp_up <- lp_signature[which(lp_signature$Average.log.fold.change > 0), ]
lp_down <- lp_signature[which(lp_signature$Average.log.fold.change < 0), ]
idx_lp_up <- match(lp_up$Symbol, rownames(mat_ct))
idx_lp_down <- match(lp_down$Symbol, rownames(mat_ct))
#对ML、basal和LP 3个signatures基因，将上调基因的表达值减去下调基因表达值，并分别返回结果。
normsig_avg_exprs <- apply(mat_ct, 2, function(x){
avg_ml_up <- mean(x[idx_ml_up])
avg_ml_down <- mean(x[idx_ml_down])
avg_ml_both <- avg_ml_up - avg_ml_down
avg_basal_up <- mean(x[idx_basal_up])
avg_basal_down <- mean(x[idx_basal_down])
avg_basal_both <- avg_basal_up - avg_basal_down
avg_lp_up <- mean(x[idx_lp_up])
avg_lp_down <- mean(x[idx_lp_down])
avg_lp_both <- avg_lp_up - avg_lp_down
return(c(avg_ml_up, avg_basal_up, avg_lp_up, avg_ml_both, avg_basal_both, avg_lp_both))
})
rownames(normsig_avg_exprs) <- c("avg_ml_up", "avg_basal_up", "avg_lp_up", "avg_ml_both", "avg_basal_both", "avg_lp_both")
#检查数据
all.equal(colnames(normsig_avg_exprs), rownames(pd_ct))
#只看上皮细胞群
normsig_avg_exprs_epithelial <- normsig_avg_exprs[,which(pd_ct$cell_types_cl_all == "epithelial")]
normsig_avg_ups <- normsig_avg_exprs[c(1:3), ]
all.equal(colnames(normsig_avg_ups), rownames(pd_ct))
normsig_avg_ups_epithelial <- normsig_avg_ups[,which(pd_ct$cell_types_cl_all == "epithelial")]
normsig_avg_both <- normsig_avg_exprs[c(4:6),]
all.equal(colnames(normsig_avg_both), rownames(pd_ct))
normsig_avg_both_epithelial <- normsig_avg_both[,which(pd_ct$cell_types_cl_all == "epithelial")]
#挑选最大值=上调基因的平均表达值最大数值分配给对应的细胞类型
assignments_normsig_ups <- apply(normsig_avg_ups, 2, function(x){rownames(normsig_avg_ups)[which.max(x)]})
assignments_normsig_ups_epithelial <- assignments_normsig_ups[which(pd_ct$cell_types_cl_all == "epithelial")]
#上调基因的平均表达值-下调基因的平均表达值的最大数值分配给对应的细胞类型
assignments_normsig_both <- apply(normsig_avg_both, 2, function(x){rownames(normsig_avg_both)[which.max(x)]})
assignments_normsig_both_epithelial <- assignments_normsig_both[which(pd_ct$cell_types_cl_all == "epithelial")]
pd_ct_epith <- pd_ct[which(pd_ct$cell_types_cl_all == "epithelial"),]
normsig_epith_pat_both <- list()
normsig_epith_pat_ups <- list()
pds_epith_ct <- list()
for (i in 1:length(patients_now)) {
normsig_epith_pat_both[[i]] <- normsig_avg_both_epithelial[,which(pd_ct_epith$patient == patients_now[i])]
normsig_epith_pat_ups[[i]] <- normsig_avg_ups_epithelial[,which(pd_ct_epith$patient == patients_now[i])]
pds_epith_ct[[i]] <- pds_ct[[i]][which(pds_ct[[i]]$cell_types_cl_all == "epithelial"),]
}
names(normsig_epith_pat_both) <- patients_now
names(normsig_epith_pat_ups) <- patients_now
names(pds_epith_ct) <- patients_now
ht_sep_normsig_both <-
Heatmap(normsig_epith_pat_both[[1]],
col = colorRamp2(c(-0.7, -0.2, 0.7), c("blue","white", "red")),
cluster_rows = FALSE,
show_column_names = FALSE,
column_title = patients_now[1],
top_annotation = ha_lehman_epith_pat[[1]],
column_title_gp = gpar(fontsize = 12),
show_row_names = FALSE,
name = patients_now[1],
heatmap_legend_param = list(title_gp = gpar(fontsize = 9), labels_gp = gpar(fontsize = 9))) +
Heatmap(normsig_epith_pat_both[[2]],
col = colorRamp2(c(-0.7, -0.2, 0.7), c("blue","white", "red")),
cluster_rows = FALSE,
show_column_names = FALSE,
column_title = patients_now[2],
column_title_gp = gpar(fontsize = 12),
top_annotation = ha_lehman_epith_pat[[2]],
name = patients_now[2],
show_row_names = FALSE,
heatmap_legend_param = list(title_gp = gpar(fontsize = 9), labels_gp = gpar(fontsize = 9))) +
Heatmap(normsig_epith_pat_both[[3]],
col = colorRamp2(c(-0.7, -0.2, 0.7), c("blue","white", "red")),
cluster_rows = FALSE,
show_column_names = FALSE,
column_title = patients_now[3],
column_title_gp = gpar(fontsize = 12),
top_annotation = ha_lehman_epith_pat[[3]],
name = patients_now[3],
show_row_names = FALSE,
top_annotation_height = unit(c(2), "cm"),
heatmap_legend_param = list(title_gp = gpar(fontsize = 9), labels_gp = gpar(fontsize = 9))) +
Heatmap(normsig_epith_pat_both[[4]],
col = colorRamp2(c(-0.7, -0.2, 0.7), c("blue","white", "red")),
cluster_rows = FALSE,
show_column_names = FALSE,
column_title = patients_now[4],
column_title_gp = gpar(fontsize = 12),
top_annotation = ha_lehman_epith_pat[[4]],
name = patients_now[4],
show_row_names = FALSE,
top_annotation_height = unit(c(2), "cm"),
heatmap_legend_param = list(title_gp = gpar(fontsize = 9), labels_gp = gpar(fontsize = 9))) +
Heatmap(normsig_epith_pat_both[[5]],
col = colorRamp2(c(-0.7, -0.2, 0.7), c("blue","white", "red")),
cluster_rows = FALSE,
show_column_names = FALSE,
column_title = patients_now[5],
column_title_gp = gpar(fontsize = 12),
top_annotation = ha_lehman_epith_pat[[5]],
name = patients_now[5],
show_row_names = FALSE,
top_annotation_height = unit(c(2), "cm"),
heatmap_legend_param = list(title_gp = gpar(fontsize = 9), labels_gp = gpar(fontsize = 9))) +
Heatmap(normsig_epith_pat_both[[6]],
col = colorRamp2(c(-0.7, -0.2, 0.7), c("blue","white", "red")),
cluster_rows = FALSE,
row_names_side = "right",
column_title = patients_now[6],
column_title_gp = gpar(fontsize = 12),
top_annotation = ha_lehman_epith_pat[[6]],
name = patients_now[6],
show_column_names = FALSE,
top_annotation_height = unit(c(2), "cm"),
heatmap_legend_param = list(title_gp = gpar(fontsize = 9), labels_gp = gpar(fontsize = 9)))
#画fig3b.pdf
print(draw(ht_sep_normsig_both, annotation_legend_side = "bottom"))
fig3b
# 每个样本的normal signatures 数目
all.equal(colnames(HSMM_allepith_clustering), names(assignments_normsig_both_epithelial))
pData(HSMM_allepith_clustering)$assignments_normsig_both <- assignments_normsig_both_epithelial
pData(HSMM_allepith_clustering)$assignments_normsig_ups <- assignments_normsig_ups_epithelial
#画fig3f
plot_cell_clusters(HSMM_allepith_clustering, 1, 2, color = "assignments_normsig_both", cell_size = 2) + facet_wrap(~patient)
fig3f
plot_cell_clusters(HSMM_allepith_clustering, 1, 2, color = "assignments_normsig_both", cell_size = 2) + facet_wrap(~Cluster)
#检查数据
all.equal(HSMM_allepith_clustering$Cluster, clustering_allepith)
all.equal(colnames(normsig_avg_both_epithelial), colnames(HSMM_allepith_clustering))
clust_avg_normsig_both <- matrix(NA, nrow = length(unique(HSMM_allepith_clustering$Cluster)), ncol = nrow(normsig_avg_both_epithelial))
rownames(clust_avg_normsig_both) <- paste("clust", c(1:length(unique(HSMM_allepith_clustering$Cluster))), sep = "")
colnames(clust_avg_normsig_both) <- rownames(normsig_avg_both_epithelial)
#算上皮细胞群的avg_both 平均表达值，接下来还是同样的操作
for (c in 1:length(unique(HSMM_allepith_clustering$Cluster))) {
clust_avg_normsig_both[c,] <- apply(normsig_avg_both_epithelial[,which(HSMM_allepith_clustering$Cluster == c)], 1, mean)
}
clust_avg_normsig_both <- as.data.frame(clust_avg_normsig_both)
clust_avg_normsig_both$Cluster <- rownames(clust_avg_normsig_both)
clust_avg_normsig_melt <- melt(clust_avg_normsig_both, "Cluster")
#画fig3e
ggplot(clust_avg_normsig_melt, aes(Cluster, value, fill = factor(variable), color = factor(variable),
shape = factor(variable))) +
geom_point(size = 3, stroke = 1) +
scale_shape_discrete(solid = T) +
ylab("average expression of signature in cluster") +
xlab("cluster") +
ylim(c(-0.35, 0.5))
fig3e
fig3e：Clusters 2和Clusters4 强烈表达the LP signature, 而cluster 3则高表达 ML signature.
接着为了进一步探究临床相关性，作者使用了三个gene signatures，进一步探究这868个上皮细胞的特征，这868个上皮细胞真的被研究到很彻底，真的佩服，这工作量真的好大....
第一个gene signatures:70-gene prognostic signature ，该signatures最初是从对有无转移复发患者的原发肿瘤之间差异表达基因的分析中得出的，总共70个基因。
mammaprint_long <- read.table("data/mammaprint_sig_new.txt", header = TRUE, sep = "\t")
mammaprint <- apply(mammaprint_long, 2, function(x){return(intersect(x, rownames(mat_ct)))})[,1]
mammaprint_avg_exprs <- apply(mat_ct[match(mammaprint, rownames(mat_ct)),], 2, mean)
all.equal(names(mammaprint_avg_exprs), colnames(mat_ct))
mammaprint_avg_exprs <- mammaprint_avg_exprs[which(pd_ct$cell_types_cl_all == "epithelial")]
all.equal(colnames(HSMM_allepith_clustering), names(mammaprint_avg_exprs))
pData(HSMM_allepith_clustering)$mammaprint_avg_exprs <- mammaprint_avg_exprs
# 画figS13b
plot_cell_clusters(HSMM_allepith_clustering, 1, 2, color = "mammaprint_avg_exprs", cell_size = 2) + facet_wrap(~patient) +
scale_color_continuous(low = "yellow", high = "blue")
figS13b
# 画figS13a
plot_cell_clusters(HSMM_allepith_clustering, 1, 2, color = "mammaprint_avg_exprs", cell_size = 2) + facet_wrap(~Cluster) +
scale_color_continuous(low = "yellow", high = "blue")
figS13a
第二个gene signatures:49-gene metastatic burden signature.该signatures可以区分了患者来源的小鼠TNBC异种移植模型中单个循环转移细胞所产生的高转移负荷和低转移负荷，总共包括49个基因。
zenawerb_long <- read.table("data/werb_49_metastasis_sig.txt", header = TRUE, sep = "\t")
zenawerb <- apply(zenawerb_long, 2, function(x){return(intersect(x, rownames(mat_ct)))})[,1]
zenawerb_avg_exprs <- apply(mat_ct[match(zenawerb, rownames(mat_ct)),], 2, mean)
all.equal(names(zenawerb_avg_exprs), colnames(mat_ct))
zenawerb_avg_exprs <- zenawerb_avg_exprs[which(pd_ct$cell_types_cl_all == "epithelial")]
all.equal(colnames(HSMM_allepith_clustering), names(zenawerb_avg_exprs))
pData(HSMM_allepith_clustering)$zenawerb_avg_exprs <- zenawerb_avg_exprs
#画figS14b
plot_cell_clusters(HSMM_allepith_clustering, 1, 2, color = "zenawerb_avg_exprs", cell_size = 2) + facet_wrap(~patient) +
scale_color_continuous(low = "yellow", high = "blue")
figS14b
#画figS14a
plot_cell_clusters(HSMM_allepith_clustering, 1, 2, color = "zenawerb_avg_exprs", cell_size = 2) + facet_wrap(~Cluster) +
scale_color_continuous(low = "yellow", high = "blue")
figS14a
第三个gene siganatures:从接受手术前化疗治疗的原发性乳腺癌患者的残存肿瘤群体中富集的基因中获得的，包括354个基因。
artega_long <- read.table("data/artega_sig.txt", header = TRUE, sep = "\t")
artega <- apply(artega_long, 2, function(x){return(intersect(x, rownames(mat_ct)))})[,1]
artega_avg_exprs <- apply(mat_ct[match(artega, rownames(mat_ct)),], 2, mean)
all.equal(names(artega_avg_exprs), colnames(mat_ct))
artega_avg_exprs <- artega_avg_exprs[which(pd_ct$cell_types_cl_all == "epithelial")]
all.equal(colnames(HSMM_allepith_clustering), names(artega_avg_exprs))
pData(HSMM_allepith_clustering)$artega_avg_exprs <- artega_avg_exprs
画figS15a
plot_cell_clusters(HSMM_allepith_clustering, 1, 2, color = "artega_avg_exprs", cell_size = 2) + facet_wrap(~patient) +
scale_color_continuous(low = "yellow", high = "blue")
figS15a
#画figS15b
plot_cell_clusters(HSMM_allepith_clustering, 1, 2, color = "artega_avg_exprs", cell_size = 2) + facet_wrap(~Cluster) +
scale_color_continuous(low = "yellow", high = "blue")
figS15b
#将3个gene signatures的表达值合成一个数据框
prognosis_sig <- cbind(mammaprint_avg_exprs, zenawerb_avg_exprs, artega_avg_exprs)
#取行名
colnames(prognosis_sig) <- c("mammaprint", "zenawerb", "artega")
prognosis_epith_pat <- list()
for (i in 1:length(patients_now)) {
prognosis_epith_pat[[i]] <- t(prognosis_sig)[,which(pd_ct_epith$patient == patients_now[i])]
}
names(prognosis_epith_pat) <- patients_now
for (i in 1:length(patients_now)) {
print(all.equal(colnames(prognosis_epith_pat[[1]]), rownames(pds_epith_ct[[1]])))
print(all.equal(names(clusterings_sep_allepith[[1]]), colnames(prognosis_epith_pat[[1]])))
}
ht_sep_prognosis <-
Heatmap(prognosis_epith_pat[[1]],
cluster_rows = FALSE,
col = colorRamp2(c(-0.2, 0.2, 1), c("blue","white", "red")),
show_column_names = FALSE,
column_title = patients_now[1],
top_annotation = ha_lehman_epith_pat[[1]],
column_title_gp = gpar(fontsize = 12),
show_row_names = FALSE,
name = patients_now[1],
heatmap_legend_param = list(title_gp = gpar(fontsize = 9), labels_gp = gpar(fontsize = 9))) +
Heatmap(prognosis_epith_pat[[2]],
col = colorRamp2(c(-0.2, 0.2, 1), c("blue","white", "red")),
cluster_rows = FALSE,
show_column_names = FALSE,
column_title = patients_now[2],
column_title_gp = gpar(fontsize = 12),
top_annotation = ha_lehman_epith_pat[[2]],
name = patients_now[2],
show_row_names = FALSE,
heatmap_legend_param = list(title_gp = gpar(fontsize = 9), labels_gp = gpar(fontsize = 9))) +
Heatmap(prognosis_epith_pat[[3]],
col = colorRamp2(c(-0.2, 0.2, 1), c("blue","white", "red")),
cluster_rows = FALSE,
show_column_names = FALSE,
column_title = patients_now[3],
column_title_gp = gpar(fontsize = 12),
top_annotation = ha_lehman_epith_pat[[3]],
name = patients_now[3],
show_row_names = FALSE,
heatmap_legend_param = list(title_gp = gpar(fontsize = 9), labels_gp = gpar(fontsize = 9))) +
Heatmap(prognosis_epith_pat[[4]],
col = colorRamp2(c(-0.2, 0.2, 1), c("blue","white", "red")),
cluster_rows = FALSE,
show_column_names = FALSE,
column_title = patients_now[4],
column_title_gp = gpar(fontsize = 12),
top_annotation = ha_lehman_epith_pat[[4]],
name = patients_now[4],
show_row_names = FALSE,
heatmap_legend_param = list(title_gp = gpar(fontsize = 9), labels_gp = gpar(fontsize = 9))) +
Heatmap(prognosis_epith_pat[[5]],
col = colorRamp2(c(-0.2, 0.2, 1), c("blue","white", "red")),
cluster_rows = FALSE,
show_column_names = FALSE,
column_title = patients_now[5],
column_title_gp = gpar(fontsize = 12),
top_annotation = ha_lehman_epith_pat[[5]],
name = patients_now[5],
show_row_names = FALSE,
heatmap_legend_param = list(title_gp = gpar(fontsize = 9), labels_gp = gpar(fontsize = 9))) +
Heatmap(prognosis_epith_pat[[6]],
col = colorRamp2(c(-0.2, 0.2, 1), c("blue","white", "red")),
cluster_rows = FALSE,
row_names_side = "right",
column_title = patients_now[6],
column_title_gp = gpar(fontsize = 12),
top_annotation = ha_lehman_epith_pat[[6]],
name = patients_now[6],
show_column_names = FALSE,
heatmap_legend_param = list(title_gp = gpar(fontsize = 9), labels_gp = gpar(fontsize = 9)))
#画fig4a
print(draw(ht_sep_prognosis, annotation_legend_side = "right"))
fig4a
接着看这3个gene signatures在不同clusters的细胞之间的表达分布。
clust_avg_prognosis <- matrix(NA, nrow = length(unique(HSMM_allepith_clustering$Cluster)), ncol = ncol(prognosis_sig))
#列明是clusters1-5
rownames(clust_avg_prognosis) <- paste("clust", c(1:length(unique(HSMM_allepith_clustering$Cluster))), sep = "")
#行名是mammaprint、zenawerb、artega和Cluster
colnames(clust_avg_prognosis) <- colnames(prognosis_sig)
#每个clusters的三个gene signatures的平均值
for (c in 1:length(unique(HSMM_allepith_clustering$Cluster))) {
clust_avg_prognosis[c,] <- apply(prognosis_sig[which(HSMM_allepith_clustering$Cluster == c),], 2, mean)}
prognosis_sig <- as.data.frame(prognosis_sig)
all.equal(rownames(prognosis_sig), colnames(HSMM_allepith_clustering))
prognosis_sig$Cluster <- as.numeric(HSMM_allepith_clustering$Cluster)
prognosis_melt <- melt(prognosis_sig, id.vars = c("Cluster"))
prognosis_melt$value <- as.numeric(prognosis_melt$value)
prognosis_melt <- prognosis_melt %>%
filter(Cluster %in% c(2,3,4))
#画fig4b
p <- ggplot(prognosis_melt, aes(factor(Cluster), value, fill = factor(Cluster))) +
scale_fill_manual(values = c("1" = "#ee204d", "2" = "#17806d", "3" = "#b2ec5d", "4" = "#cda4de", "5" = "#1974d2"))
p + geom_violin(adjust = .5) + facet_wrap(~variable) + stat_summary(fun.y = mean, geom = "point", shape = 22, size = 3)
fig4b
#画figS16
venn(list("PS" = mammaprint, "MBS" = zenawerb, "RTS" = artega))
figS16
可以看到这3个gene signatures没有重叠的基因，并且它们来源不同，但这3个 gene signatures均在clusters 2亚群中都高表达。接着，作者想跟临床预后联系到一起，于是选取了3个有代表性的数据集gene signature，对868个上皮细胞的5个clusters进行探索，结果提示clusters2具有与其他clusters不同的特征，接着对clusters2进行进一步探索。在之前的第五篇笔记tSNE中，已经用differentialGeneTest对每一个上皮细胞cluster与全部的上皮细胞进行差异分析后，以FDR=0.1为阈值，选取前10个有显著差异变化的基因在METABRIC 数据库中进行生存分析，作者在补充材料里已经描述得很详细。
image-20210209084756920.png
结果显示高表达clusters2的肿瘤与OS显著负相关相关性。相反，但是在三个gene signatures中的预后则无统计学意义。此外，clusters1、3、4和5的基因无法预测临床结果。这些发现揭示了单细胞测序分析能揭示与临床预后相关细胞状态的能力，但这些细胞状态还没有在肿瘤真实世界样本中没被发现。
image-20210124111022195.png
我们继续探索。在fig3a中，我们已经对868个上皮细胞进行tSNE,接着我们将这个tSNE结果，进行进一步注释可并可视化。
image-20210209091113221.png
## 可视化normal signatures，figS10b
plot_cell_clusters(HSMM_allepith_clustering, 1, 2, color = "assignments_normsig_both", cell_size = 3)
figS10b
#可视化TNBCtype4 Lehman signatures
#去掉immunomodulatory和mesenchymal_stem_like上皮细胞
lehman_avg_both_epithelial_new <- lehman_avg_both_epithelial[-which(rownames(lehman_avg_both_epithelial) %in% c("immunomodulatory", "mesenchymal_stem_like")),]
#给868个上皮细胞进行分类（basal_like_1,basal_like_2,mesenchymal,luminal_ar）
assignments_lehman_both_epithelial_new <- apply(lehman_avg_both_epithelial_new, 2, function(x){rownames(lehman_avg_both_epithelial_new)[which.max(x)]})
all.equal(colnames(HSMM_allepith_clustering), names(assignments_lehman_both_epithelial_new))
pData(HSMM_allepith_clustering)$assignments_lehman_both_new <- assignments_lehman_both_epithelial_new
#画figS10c
plot_cell_clusters(HSMM_allepith_clustering, 1, 2, color = "assignments_lehman_both_new", cell_size = 3)
figS10c
#可视化mammaprint signature (PS)
plot_cell_clusters(HSMM_allepith_clustering, 1, 2, color = "mammaprint_avg_exprs", cell_size = 3) +
scale_color_continuous(low = "yellow", high = "blue")
figS10d
然后，通过研究在cluster2中与其他上皮细胞群的差异表达的基因中富集的通路变化丰，来确定cluster2亚群背后的功能机制。我们发现，最富集的通路与鞘糖脂的生物合成和溶酶体的周转有关，这两种途径影响了同样富集到的先天免疫系统的细胞因子途径。接着可视化每个样本的鞘糖脂的生物合成和先天免疫系统的细胞因子两条途径的热图。
糖鞘糖脂最近被认为是乳腺癌中许多促进肿瘤特性的介质，包括改变的生长因子信号、EMT和干样行为。该途径中的多个关键基因在cluster2亚群中高表达，包括糖脂转移蛋白基因GLTP和鞘脂生物合成关键亚单位基因SPTLC1。鞘磷脂也被认为是天然免疫系统和获得性免疫系统的重要调节剂，并与多种上皮组织炎症相关癌变有关。
#path1是糖鞘糖脂的关键基因
path1 <- c("ST3GAL4", "ST3GAL6", "ST8SIA1", "FUT1", "FUT2", "FUT3", "FUT4", "FUT6", "FUT9", "GLTP", "SPTLC1", "KDSR", "SPTLC2", "CERK", "ARSA")
idx_path1 <- match(path1, rownames(HSMM_allepith_clustering))
#path1是先天免疫系统的细胞因子途径的关键基因
path2 <- c("CCL20", "CCL22", "CCL4", "CCR6", "IL11", "IL12RB1", "IL6R", "CSF2RA", "BMP7", "GLMN", "GPI", "INHBA", "TNF", "TNFSF15", "GHR", "LEPR", "TLR1", "TLR2", "TLR5", "TLR7", "TLR10", "F11R")
idx_path2 <- match(path2, rownames(HSMM_allepith_clustering))
path3 <- c("ERBB2", "AKT1", "AKT3", "PIK3R3", "PIK3R4", "RPS6KB2", "TRIB3", "BTK", "GRB10", "GRB2", "ILK", "PAK1", "PRKCZ", "CSNK2A1",
"IRS1", "IRAK1", "MYD88", "MAP2K1", "MAPK8", "MAPK1", "PTPN11", "EIF4E", "EIF4EBP1", "EIF4G1", "FKBP1A", "PDK1", "RHEB", "RPS6KB1")
idx_path3 <- match(path3, rownames(HSMM_allepith_clustering))
all_idx_paths <- c(idx_path1, idx_path2)
#命名
names_path <- c(rep("glycosphigolipid metabolism", length(idx_path1)), rep("innate immunity", length(idx_path2)))
#上色
anno_cols_path <- c("glycosphigolipid metabolism" = "#ff9baa", "innate immunity" = "#17806d")
ha_path_rows <- HeatmapAnnotation(df = data.frame(pathway = names_path),
annotation_legend_param = list(pathway = list(ncol = 1, title = "pathway", title_position = "topcenter")),
which = "row", col = list("pathway" = anno_cols_path), annotation_width = unit(3, "mm"))
#分离每个cluster的矩阵，并提取关键基因。
mat_epith_allpats <- exprs(HSMM_allepith_clustering)
mats_epith_patient <- list()
pds_epith_patient <- list()
mats_epith_patient_clusters <- list()
for (i in 1:length(patients_now)) {
pds_epith_patient[[i]] <- pData(HSMM_allepith_clustering)[which(pData(HSMM_allepith_clustering)$patient == patients_now[i]), ]
mats_epith_patient[[i]] <- mat_epith_allpats[all_idx_paths, which(pData(HSMM_allepith_clustering)$patient == patients_now[i])]
mats_epith_patient_clusters[[i]] <- list()
for (c in 1:length(unique(HSMM_allepith_clustering$Cluster))) {
mats_epith_patient_clusters[[i]][[c]] <- mats_epith_patient[[i]][, which(pds_epith_patient[[i]]$Cluster == c)]
}
names(mats_epith_patient_clusters[[i]]) <- paste0("clust", c(1:5))
}
names(mats_epith_patient) <- patients_now
names(pds_epith_patient) <- patients_now
# 接下来就是画图了。
ht_paths <-
ha_path_rows +
Heatmap(mats_epith_patient_clusters[[1]][[1]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
show_column_dend = TRUE, show_column_names = FALSE,
name = "cluster1", column_title = "cluster1",
row_names_gp = gpar(fontsize = 9, col = anno_cols_path),
split = names_path) +
Heatmap(mats_epith_patient_clusters[[1]][[2]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster2", column_title = "cluster2",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[1]][[3]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster3", column_title = "cluster3",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[1]][[4]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster4", column_title = "cluster4",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[1]][[5]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster5", column_title = "cluster5",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[2]][[1]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
show_column_dend = TRUE, show_column_names = FALSE,
name = "cluster1_2", column_title = "cluster1",
row_names_gp = gpar(fontsize = 9, col = anno_cols_path),
split = names_path) +
Heatmap(mats_epith_patient_clusters[[2]][[2]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster2_2", column_title = "cluster2",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[2]][[3]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster3_2", column_title = "cluster3",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[2]][[4]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster4_2", column_title = "cluster4",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[2]][[5]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster5_2", column_title = "cluster5",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[3]][[1]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster1_3", column_title = "cluster1",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[3]][[2]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster2_3", column_title = "cluster2",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[3]][[3]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster3_3", column_title = "cluster3",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[3]][[4]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster4_3", column_title = "cluster4",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[3]][[5]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster5_3", column_title = "cluster5",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[4]][[1]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster1_4", column_title = "cluster1",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[4]][[2]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster2_4", column_title = "cluster2",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[4]][[3]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster3_4", column_title = "cluster3",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[4]][[4]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster4_4", column_title = "cluster4",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[4]][[5]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster5_4", column_title = "cluster5",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[5]][[1]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster1_5", column_title = "cluster1",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[5]][[2]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster2_5", column_title = "cluster2",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[5]][[3]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster3_5", column_title = "cluster3",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[5]][[4]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster4_5", column_title = "cluster4",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[5]][[5]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster5_5", column_title = "cluster5",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[6]][[1]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster1_6", column_title = "cluster1",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[6]][[2]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster2_6", column_title = "cluster2",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[6]][[3]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster3_6", column_title = "cluster3",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[6]][[4]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster4_6", column_title = "cluster4",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE) +
Heatmap(mats_epith_patient_clusters[[6]][[5]],
col = colorRamp2(c(-0.5, 1, 3), c("blue", "white", "red")),
cluster_rows = TRUE, show_row_dend = FALSE,
name = "cluster5_6", column_title = "cluster5",
show_row_names = FALSE,
show_column_dend = TRUE, show_column_names = FALSE)
fig4d
画得有点丑，画图参数得调整需要参考ComplexHeatmap包的说明书，继续美化一下。
到了这里，这篇文献的单细胞部分就学习完了，花了一个月的时间断断续续把这个系列共6篇笔记写完。作者把这1000多个细胞不断分群，不断探索，最后集中在了上皮细胞群，进一步细分，然后结合临床预后情况，继续探索。这是很标准的单细胞分析流程。当然，文章中也加了一些免疫组化的实验方法，加以验证。还有一部分外显子测序，这又是另外一个需要学习的领域了，在单细胞学习的路上，感恩遇到得到了“单细胞天地”公众号团队的答疑解惑。2021年，希望能学到更多的东西，写更多的笔记，相信一切的努力都不会白白付出。
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