安装软件
wget -O cellranger-6.0.2.tar.gz "https://cf.10xgenomics.com/releases/cell-exp/cellranger-6.0.2.tar.gz?Expires=1626276507&Policy=eyJTdGF0ZW1lbnQiOlt7IlJlc291cmNlIjoiaHR0cHM6Ly9jZi4xMHhnZW5vbWljcy5jb20vcmVsZWFzZXMvY2VsbC1leHAvY2VsbHJhbmdlci02LjAuMi50YXIuZ3oiLCJDb25kaXRpb24iOnsiRGF0ZUxlc3NUaGFuIjp7IkFXUzpFcG9jaFRpbWUiOjE2MjYyNzY1MDd9fX1dfQ__&Signature=lA3CN57Qf2GAcEJT3FXW-g92R1IzJEosRg0EZxpLGfR5JpvgT9zK9kzroDLCNGQj1ov-CMNABfjMqsFisMHZW8S-tHg1Qv6muqiLvkd~04XEpJEH3TYKRsziiEFhvlZxpd-XvnOTTODtSYkcVcNGhUaGzC-jdy7Q~RR-oGp~gkllK6gFdIXo4sWrbbSogNv8X2y6bsWHMATXkEnP2f0Bub5mnE88w1hysiyub3-FqOFjYO1tAUqwcnAdrLn8jJjjlSt1emW5NANgCOdW-Fw4o5N0Bu6xJwrXAdzIey3JcJc6GnLgSUoselPlJt3zNU0dreBjckP6ERWI~jMXPQ0fPQ__&Key-Pair-Id=APKAI7S6A5RYOXBWRPDA"
#解压缩
tar -xzvf cellranger-6.0.2.tar.gz
下载人的参考基因组 10×官网给定的
wget https://cf.10xgenomics.com/supp/cell-exp/refdata-gex-GRCh38-2020-A.tar.gz
tar -xzvf refdata-gex-GRCh38-2020-A.tar.gz
添加软件目录到环境中
vi ~/.bashrc
export PATH="/public/vip/biosoft/cellranger/cellranger-6.0.2:$PATH"
source ~/.bashrc
运行测试
cellranger
cellranger cellranger-6.0.2
Process 10x Genomics Gene Expression, Feature Barcode, and Immune Profiling data
USAGE:
cellranger <SUBCOMMAND>
FLAGS:
-h, --help Prints help information
-V, --version Prints version information
SUBCOMMANDS:
count Count gene expression (targeted or whole-transcriptome) and/or feature barcode
reads from a single sample and GEM well
multi Analyze multiplexed data or combined gene expression/immune profiling/feature
barcode data
vdj Assembles single-cell VDJ receptor sequences from 10x Immune Profiling libraries
aggr Aggregate data from multiple Cell Ranger runs
reanalyze Re-run secondary analysis (dimensionality reduction, clustering, etc)
targeted-compare Analyze targeted enrichment performance by comparing a targeted sample to its
cognate parent WTA sample (used as input for targeted gene expression)
targeted-depth Estimate targeted read depth values (mean reads per cell) for a specified input
parent WTA sample and a target panel CSV file
mkvdjref Prepare a reference for use with CellRanger VDJ
mkfastq Run Illumina demultiplexer on sample sheets that contain 10x-specific sample index
sets
testrun Execute the 'count' pipeline on a small test dataset
mat2csv Convert a gene count matrix to CSV format
mkref Prepare a reference for use with 10x analysis software. Requires a GTF and FASTA
mkgtf Filter a GTF file by attribute prior to creating a 10x reference
upload Upload analysis logs to 10x Genomics support
sitecheck Collect linux system configuration information
help Prints this message or the help of the given subcommand(s)
https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest
https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/installation
数据下载
wget https://cg.10xgenomics.com/samples/cell-exp/3.0.0/pbmc_10k_v3/pbmc_10k_v3_fastqs.tar &
ls -hlt
total 49G
-rw-r--r-- 1 vip vip 16G 11月 2 2018 pbmc_10k_v3_S1_L001_R2_001.fastq.gz
-rw-r--r-- 1 vip vip 16G 11月 2 2018 pbmc_10k_v3_S1_L002_R2_001.fastq.gz
-rw-r--r-- 1 vip vip 6.6G 11月 2 2018 pbmc_10k_v3_S1_L001_R1_001.fastq.gz
-rw-r--r-- 1 vip vip 6.6G 11月 2 2018 pbmc_10k_v3_S1_L002_R1_001.fastq.gz
-rw-r--r-- 1 vip vip 2.1G 11月 2 2018 pbmc_10k_v3_S1_L001_I1_001.fastq.gz
-rw-r--r-- 1 vip vip 2.1G 11月 2 2018 pbmc_10k_v3_S1_L002_I1_001.fastq.gz
nohup wget https://cf.10xgenomics.com/samples/cell-exp/3.0.0/pbmc_1k_v3/pbmc_1k_v3_fastqs.tar &
ls -hlt
total 5.2G
-rw-r--r-- 1 vip vip 1.7G 11月 2 2018 pbmc_1k_v3_S1_L001_R2_001.fastq.gz
-rw-r--r-- 1 vip vip 1.7G 11月 2 2018 pbmc_1k_v3_S1_L002_R2_001.fastq.gz
-rw-r--r-- 1 vip vip 719M 11月 2 2018 pbmc_1k_v3_S1_L001_R1_001.fastq.gz
-rw-r--r-- 1 vip vip 714M 11月 2 2018 pbmc_1k_v3_S1_L002_R1_001.fastq.gz
-rw-r--r-- 1 vip vip 245M 11月 2 2018 pbmc_1k_v3_S1_L001_I1_001.fastq.gz
-rw-r--r-- 1 vip vip 243M 11月 2 2018 pbmc_1k_v3_S1_L002_I1_001.fastq.gz</pre>
运行
cellranger count --id=run_count_1kpbmcs --fastqs=/public/vip/project/sc_10X_RNA_project/pbmc_example_data/pbmc_1k_v3_fastqs --sample=pbmc_1k_v3 --transcriptome=/public/vip/reference/sc_10X_RNA_reference/refdata-gex-GRCh38-2020-A
输出结果
ls -hlt
total 72K
drwxrwxr-x 4 vip vip 4.0K 7月 14 18:16 run_count_1kpbmcs
-rw------- 1 vip vip 61K 7月 14 18:16 nohup.out
进入文件夹查看
ls -hlt
total 5.5M
-rw-rw-r-- 1 vip vip 2.5M 7月 14 18:16 run_count_1kpbmcs.mri.tgz
-rw-rw-r-- 1 vip vip 114K 7月 14 18:16 _filelist
-rw-rw-r-- 1 vip vip 18K 7月 14 18:16 _sitecheck
-rw-rw-r-- 1 vip vip 229 7月 14 18:16 _cmdline
-rw-r--r-- 1 vip vip 89K 7月 14 18:16 _log
-rw-r--r-- 1 vip vip 1.8M 7月 14 18:16 _finalstate
-rw-r--r-- 1 vip vip 693K 7月 14 18:16 _perf
-rw-r--r-- 1 vip vip 51 7月 14 18:16 _timestamp
drwxrwxr-x 5 vip vip 4.0K 7月 14 18:16 outs
-rw-r--r-- 1 vip vip 57K 7月 14 18:16 _vdrkill
drwxrwxr-x 9 vip vip 4.0K 7月 14 16:58 SC_RNA_COUNTER_CS
-rw-r--r-- 1 vip vip 1.2K 7月 14 16:57 _invocation
-rw-r--r-- 1 vip vip 5 7月 14 16:57 _jobmode
-rw-r--r-- 1 vip vip 228K 7月 14 16:57 _mrosource
-rw-r--r-- 1 vip vip 2 7月 14 16:57 _tags
-rw-r--r-- 1 vip vip 36 7月 14 16:57 _uuid
-rw-r--r-- 1 vip vip 64 7月 14 16:57 _versions
主要文件存放在这个文件夹里
cd outs/
ls -hlt
total 4.1G
-rw-rw-r-- 2 vip vip 21M 7月 14 18:16 cloupe.cloupe
-rw-rw-r-- 1 vip vip 3.4M 7月 14 18:15 web_summary.html
-rw-rw-r-- 1 vip vip 650 7月 14 18:15 metrics_summary.csv
drwxrwxr-x 7 vip vip 4.0K 7月 14 18:13 analysis
-rw-rw-r-- 1 vip vip 44M 7月 14 18:08 molecule_info.h5
drwxrwxr-x 2 vip vip 4.0K 7月 14 18:08 filtered_feature_bc_matrix
-rw-rw-r-- 1 vip vip 4.0M 7月 14 18:08 filtered_feature_bc_matrix.h5
-rw-rw-r-- 1 vip vip 3.9M 7月 14 18:07 possorted_genome_bam.bam.bai
-rw-rw-r-- 1 vip vip 4.0G 7月 14 18:06 possorted_genome_bam.bam
drwxrwxr-x 2 vip vip 4.0K 7月 14 18:02 raw_feature_bc_matrix
-rw-rw-r-- 1 vip vip 8.1M 7月 14 18:01 raw_feature_bc_matrix.h5
