10X空间转录组数据分析之方法小结

今天我们来提炼一下10X空间转录组在文章中一些新的运用，文献在The cellular architecture of the antimicrobial response network in human leprosy granulomas,2021年6月发表于nature immunology，IF26分。

这篇文章前面了做了很多单细胞方面的内容，其中对于细胞类型的识别尤为细致，做有关研究的同学可以借鉴一下其中的marker gene和思路。

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单细胞其实做了大量的基础工作，非常耗时间和精力，给作者点个赞。单细胞这里给大家强调一点就是基因网络的构建。

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• 注：Circos plots showing the connection between IL1B (left) or IFNG (right) and the direct antimicrobial gene targets in the cell types with a z-score > 3. The color represents the patient composition of the cell type: red, L-lep-specific; blue, RR-specific; gray, mix of L-lep and RR.

接下来就是我们的重点方向，空间转录组

1、空间转录组的注释，注意这里的注释方式。

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依据形态学的位置注释，但更为精细，Spatial plot for 708 spots colored by clusters; the coordinates of the spot correspond to the location in the tissue。当然，划分好区域之后，很多新奇的发现就产生了。

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2、空间转录组的聚类方式

单细胞空间联合分析后的结果， The predicted cell type composition for each spot was then used to cluster the spots by the k-means algorithm. The clusters were annotated based on the average cell type prediction score across all the spots in the cluster.也就是按照细胞类型注释的矩阵进行聚类，这个我之前分享的文章提到过，非常重要，大家可以参考10X单细胞-10X空间转录组联合分析之八----STRIDE（三维重构）。

3、空间位置距离，这个不止一次提到过，希望引起大家的重视

We reasoned that adjacency in spatial location would facilitate cell–cell interactions（临近的细胞才有相互作用）. By quantifying the neighboring spots, as shown in the scatter plot

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we found that in T-lep/RR#samples, myeloid cells were most adjacent to T cells, keratinocytes were adjacent to fibroblasts and fibroblasts were equidistant from all cell types

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• 注：Heatmap showing the number of neighbors for each pair of cell types in the RR and T-lep spatial-seq samples. The percentage of the numbers were calculated for each row, thus each row sum to 1.

The results from the two L-lep samples indicated that the immune, myeloid, plasma and T cells were close to each other, with fibroblasts being close to the myeloid cells。（位置关系非常重要，希望大家能重视起来）。

细胞之间的距离度量

To measure the distance from fibroblast subtypes to the epidermis, the FB0, FB2 and epidermis spots were first defined（首先定义空间位点）. Epidermis spots were defined based on the expression of KRT1 and KRT10. Spots with higher expression than the 90% quantile of the sum of these two genes were considered epidermis spots(这个方式也是重点). FB0 and FB2 spots were defined using the subtype prediction scores. Spots that had FB0 prediction scores higher than the 90% quantile were considered FB0 spots and spots that had FB2 prediction scores higher than the 90% quantile were considered FB2 spots(非常重要). The locations of the spots were extracted from the ‘tissue_positions_list.csv’ file from the spaceranger output. The Euclidean distance from the FB0 and FB2 spots to all the epidermis spots were calculated and the shortest distance was used as the distance from the spot to the epidermis. All three RR samples and the T-lep samples were combined and a Wilcoxon rank-sum test was performed between the FB0 epidermis and FB2 epidermis distances.(这个地方的可操作性很强，很值得大家学习)。

临近矩阵分析（一般是做细胞临近通讯的重点，也是细胞之间interaction的重点）

For each spot, the closed six spots surrounding it were considered nearest neighbors. The number of neighboring spots for each pair of cell types were counted in each of the spatial sequencing samples to generate a nearest-neighbor matrix. Each row of the matrix was divided by the row sum.

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生活很好，有你更好