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mAGLAVE edited this page Jun 22, 2021
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Perform single-cell RNA-seq analysis from FastQ files to cerebro file for 10XGenomics technology data.
Individual Analysis
- Alignments (Alignment_countTable_GE, Alignment_countTable_ADT, Alignment_annotations_TCR_BCR),
- QC of Droplets and filetring (Droplets_QC_GE, Filtering_GE),
- Normalization and dimension Reduction (Norm_DimRed_Eval_GE),
- Clustering, Identification of Marker Genes and Annotation of clusters (Clust_Markers_Annot_GE),
- Integration of several additional "omics" (Adding_ADT, Adding_TCR, Adding_BCR),
- Creation of a Cerebro object to help vizualisation of results (Cerebro).
Integrated Analysis of several samples
- Integration, Normalization and dimension Reduction (Int_Norm_DimRed_Eval_GE)
- Clustering, Identification of Marker Genes and Annotation of clusters (Int_Clust_Markers_Annot_GE),
- Integration of several additional "omics" (Int_Adding_ADT, Int_Adding_TCR, Int_Adding_BCR),
- Creation of a Cerebro object to help vizualisation of results (Cerebro).
Grouped Analysis (no integration) of several samples
- Merger, Normalization and dimension Reduction (Grp_Norm_DimRed_Eval_GE)
- Clustering, Identification of Marker Genes and Annotation of clusters (Grp_Clust_Markers_Annot_GE),
- Integration of several additional "omics" (Grp_Adding_ADT, Grp_Adding_TCR, Grp_Adding_BCR),
- Creation of a Cerebro object to help vizualisation of results (Cerebro).
- Improve cell annotation,
- Update to CellRanger 6.0.0,
- Analysis of scATAC-seq,
- ...
![wiki_sidebar](https://github.com/gustaveroussy/single-cell/raw/master/images/wiki_sidebar.png)
Resources of the Theory of single cell RNA-seq
v1.3
Pipeline details
Configuration
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Parameter file
- Steps
- Alignment_countTable_GE
- Droplets_QC_GE
- Filtering_GE
- Norm_DimRed_Eval_GE
- Clust_Markers_Annot_GE
- Cerebro
- Alignment_countTable_ADT
- Adding_ADT
- Alignment_annotations_TCR_BCR
- Adding_TCR
- Adding_BCR
- Int_Norm_DimRed_Eval_GE
- Int_Clust_Markers_Annot_GE
- Int_Adding_ADT
- Int_Adding_TCR
- Int_Adding_BCR
- Grp_Norm_DimRed_Eval_GE
- Grp_Clust_Markers_Annot_GE
- Grp_Adding_ADT
- Grp_Adding_TCR
- Grp_Adding_BCR
- Additional files
Results help
- Arborescence of all results
-
Observations and weird results
- Not a threshold by emptyDrops
- Large and small cells into the same sample
- emptyDrops does't work well
- More than 15% mitochondrial RNA while I filtered them out at 15%
- Impact of empty droplets on umap
- Choose the right number of dimensions
- Be careful with the colors, they are sometimes misleading
- Impact of bias correction on umap
Complete Examples of school cases
Individual analysis :
1 sample (scRNA-seq + ADT + TCR + BCR)
Grouped/Integrated analysis :
2 samples (scRNA-seq + ADT + TCR + BCR)