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10x Genomics
Chromium Single Cell Gene Expression

Cell Ranger3.0, printed on 11/21/2024

Run Analysis

The count, aggr and reanalyze pipelines output several CSV files which contain automated secondary analysis results. A subset of these results are used to render the Analysis View in the run summary.

Dimensionality Reduction

Before clustering the cells, Principal Component Analysis (PCA) is run on the normalized filtered feature-barcode matrix to reduce the number of feature (gene) dimensions. Only gene expression features are used as PCA features. The PCA analysis produces four output files. The first is a projection of each cell onto the first N principal components. By default N=10 (N=100 when chemistry batch correction is enabled); when running reanalyze, you can choose to increase it.

$ cd /home/jdoe/runs/sample345/outs
$ head -2 analysis/pca/10_components/projection.csv
Barcode,PC-1,PC-2,PC-3,PC-4,PC-5,PC-6,PC-7,PC-8,PC-9,PC-10
AAACATACAACGAA-1,-0.2765,-5.7056,6.5324,-12.2736,-1.4390,-1.1656,-0.1754,-2.9748,3.3785,1.6539

The second file is a components matrix which indicates how much each feature contributed (the loadings) to each principal component. Features that were not included in the PCA analysis have all of their loading values set to zero.

$ head -2 analysis/pca/10_components/components.csv
PC,ENSG00000228327,ENSG00000237491,ENSG00000177757,ENSG00000225880,...,ENSG00000160310
1,-0.0044,0.0039,-0.0024,-0.0016,...,-0.0104

The third file records the proportion of total variance explained by each principal component. When choosing the number of principal components that are significant, it is useful to look at the plot of variance explained as a function of PC rank - when the numbers start to flatten out, subsequent PCs are unlikely to represent meaningful variation in the data.

$ head -5 analysis/pca/10_components/variance.csv
PC,Proportion.Variance.Explained
1,0.0056404970744118104
2,0.0038897311237809061
3,0.0028803714818085419
4,0.0020830581822081206

The final file lists the normalized dispersion of each feature, after binning features by their mean expression across the dataset. This provides a useful measure of variability of each feature.

$ head -5 analysis/pca/10_components/dispersion.csv
Feature,Normalized.Dispersion
ENSG00000228327,2.0138970131886671
ENSG00000237491,1.3773662040549017
ENSG00000177757,-0.28102027567224191
ENSG00000225880,1.9887312950109921

t-SNE

After running PCA, t-distributed Stochastic Neighbor Embedding (t-SNE) is run to visualize cells in a 2-D space.

$ head -5 analysis/tsne/2_components/projection.csv
Barcode,TSNE-1,TSNE-2
AAACATACAACGAA-1,-13.5494,1.4674
AAACATACTACGCA-1,-2.7325,-10.6347
AAACCGTGTCTCGC-1,12.9590,-1.6369
AAACGCACAACCAC-1,-9.3585,-6.7300

Clustering

Clustering is then run to group cells together that have similar expression profiles, based on their projection into PCA space. Graph-based clustering (under graphclust) is run once as it does not require prespecification of the number of clusters. K-means (under kmeans) is run for many values of K=2,...,N where K corresponds to the number of clusters. By default N=10; when running reanalyze, you can choose to increase it. The corresponding results for each K is separated into its own directory.

$ ls analysis/clustering
graphclust         kmeans_10_clusters  kmeans_2_clusters  kmeans_3_clusters
kmeans_4_clusters  kmeans_5_clusters   kmeans_6_clusters  kmeans_7_clusters
kmeans_8_clusters  kmeans_9_clusters

For each clustering, cellranger produces cluster assignments for each cell.

$ head -5 analysis/clustering/kmeans_3_clusters/clusters.csv
Barcode,Cluster
AAACATACAACGAA-1,2
AAACATACTACGCA-1,2
AAACCGTGTCTCGC-1,1
AAACGCACAACCAC-1,3

Differential Expression

cellranger also produces a table indicating which features are differentially expressed in each cluster relative to all other clusters. For each feature we compute three values per cluster:

This is located in a different directory than the clustering results, but follows the same structure, with each clustering separated into its own directory.

$ head -5 analysis/diffexp/kmeans_3_clusters/differential_expression.csv

Feature ID,Feature Name,Cluster 1 Mean UMI Counts,Cluster 1 Log2 fold change,Cluster 1 Adjusted p value,Cluster 2 Mean UMI Counts,Cluster 2 Log2 fold change,Cluster 2 Adjusted p value,Cluster 3 Mean UMI Counts,Cluster 3 Log2 fold change,Cluster 3 Adjusted p value
ENSG00000228327,RP11-206L10.2,0.0056858989363338264,2.6207666981569986,0.00052155805898912184,0.0,-0.75299726644507814,0.64066099091888962,0.00071455453829430329,-2.3725403666493312,0.0043023680184636837
ENSG00000237491,RP11-206L10.9,0.00012635330969630726,-0.31783275717885928,0.40959138980118809,0.0,3.8319652342760779,0.11986963938734894,0.0,0.56605908868652577,0.39910771338768203
ENSG00000177757,FAM87B,0.0,-2.9027952579000154,0.0,0.0,3.2470027335549219,0.19129034227967889,0.00071455453829430329,3.1510215894076818,0.0
ENSG00000225880,LINC00115,0.0003790599290889218,-5.71015017995762,8.4751637615375386e-28,0.20790015775229512,7.965820981010868,1.3374521290889345e-46,0.0017863863457357582,-2.2065304152104019,0.00059189960914085744

Multiple Species

If you analyzed a multi-species experiment, the analysis output will look different. For example, the human-mouse mixing experiment is run to verify system functionality. It consists of mixing approximately 600 human (HEK293T) cells and 600 mouse (3T3) cells in a 1:1 ratio.

cellranger produces a single analysis CSV file indicating whether each GEM contains only a single human cell (hg19), a single mouse cell (mm10) or multiple mouse and human cells (Multiplet).

$ cd /home/jdoe/runs/sample345/outs
$ head -5 analysis/gem_classification.csv
barcode,hg19,mm10,call
AAACATACACCTCC-1,3,815,mm10
AAACATACACCTGA-1,14,780,mm10
AAACATACACGTGT-1,2,439,mm10
AAACATACAGACTC-1,700,776,Multiplet