Single Cell Gene Expression Pipeline: Cell Ranger
FIXME: intro
Getting set up
The basic method for logging on to rhino and requesting computational resources to run cellranger are as follows:
- Log on to rhino:
ssh username@rhino - Grab a node:
grabnode - Load software:
ml cellranger
For in-depth descriptions of each step, please see this tutorial in the FH Biomedical Data Science Wiki.
This material below is modified from an original tutorial from 10x Genomics available here.
Project organization
FIXME
mkdir ~/yard/run_cellranger_mkfastq
cd ~/yard/run_cellranger_mkfastq
Downloading data
other data upload options here
FIXME
wget http://cf.10xgenomics.com/supp/cell-exp/cellranger-tiny-bcl-1.2.0.tar.gz
wget http://cf.10xgenomics.com/supp/cell-exp/cellranger-tiny-bcl-simple-1.2.0.csv
tar -zxvf cellranger-tiny-bcl-1.2.0.tar.gz
cat cellranger-tiny-bcl-simple-1.2.0.csv
tree -L 2 cellranger-tiny-bcl-1.2.0
Running Cell Ranger
FIXME: about Cell Ranger
Demultiplexing Illumina base call files (BCL) with cellranger mkfastq
FIXME
cellranger mkfastq --help
FIXME
cellranger mkfastq --id=tutorial_walk_through \
--run=cellranger-tiny-bcl-1.2.0 \
--csv=cellranger-tiny-bcl-simple-1.2.0.csv
Align sequence reads to reference using cellranger count
mkdir ~/yard/run_cellranger_count
cd ~/yard/run_cellranger_count
wget http://cf.10xgenomics.com/samples/cell-exp/3.0.0/pbmc_1k_v3/pbmc_1k_v3_fastqs.tar
tar -xvf pbmc_1k_v3_fastqs.tar
wget http://cf.10xgenomics.com/supp/cell-exp/refdata-cellranger-GRCh38-3.0.0.tar.gz
tar -zxvf refdata-cellranger-GRCh38-3.0.0.tar.gz
cellranger count --help
cellranger count --id=run_count_1kpbmcs \
--fastqs=run_cellranger_count/pbmc_1k_v3_fastqs \
--sample=pbmc_1k_v3 \
--transcriptome=run_cellranger_count/refdata-cellranger-GRCh38-3.0.0
Aggregate samples using cellranger aggr
cellranger aggr --help
mkdir ~/yard/run_cellranger_aggr
cd ~/yard/run_cellranger_aggr
wget http://cf.10xgenomics.com/samples/cell-exp/3.0.0/pbmc_1k_v3/pbmc_1k_v3_molecule_info.h5
wget http://cf.10xgenomics.com/samples/cell-exp/3.0.0/pbmc_10k_v3/pbmc_10k_v3_molecule_info.h5
pwd
Copy the output of the command above, use to create csv below
nano pbmc_aggr.csv
cellranger aggr --id=1k_10K_pbmc_aggr --csv=pbmc_aggr.csv
ls -1 1k_10K_pbmc_aggr/outs/
Rerun an analysis with different parameters using cellranger reanalyze
mkdir ~/yard/run_cellranger_reanalyze
cd ~/yard/run_cellranger_reanalyze
cellranger reanalyze --help
wget http://cf.10xgenomics.com/samples/cell-exp/3.0.0/pbmc_10k_v3/pbmc_10k_v3_filtered_feature_bc_matrix.h5
create parameters file:
nano reanalyze_10k_pbmcs.csv
num_principal_comps,14
max_clusters,15
cellranger reanalyze --id=10k_pbmc_reanalyze_pc_clust --matrix=pbmc_10k_v3_filtered_feature_bc_matrix.h5 --params=reanalyze_10k_pbmcs.csv
ls -1 10k_pbmc_reanalyze_pc_clust/outs/