幫助文檔地址
總流程
Install the Cell Ranger Pipeline
Check the system to see if it meets the system requirements
Use cellranger upload to send data to 10X Genomics support
Know where to get help
Run cellranger mkfastq to generate FASTQ files using test data
Run cellranger count using a public data set
Run cellranger aggr to combine two data sets
Run cellranger reanalyze to adjust clustering parameters
cellranger count 流程
- 得到fastq和參考基因組文件
cellranger count是通過(guò)將fastq序列比對(duì)到參考轉(zhuǎn)錄組上并產(chǎn)生一個(gè)以.cloupe為結(jié)尾的文件以便在loupe cell browser上分析，同時(shí)會(huì)產(chǎn)生多個(gè)與目前主流分析軟件兼容的文件以便進(jìn)一步分析
mkdir ~/yard/run_cellranger_count
cd ~/yard/run_cellranger_count
本例中以一個(gè)1000個(gè)細(xì)胞的PBMC的數(shù)據(jù)集為例作為演示螟蒸，首先下載該數(shù)據(jù)集
wget http://cf.10xgenomics.com/samples/cell-exp/3.0.0/pbmc_1k_v3/pbmc_1k_v3_fastqs.tar
該文件大概為5.17G鱼填，文件下載后要解壓
tar -xvf pbmc_1k_v3_fastqs.tar
解壓后的文件列表如下
pbmc_1k_v3_fastqs/
pbmc_1k_v3_fastqs/pbmc_1k_v3_S1_L001_R2_001.fastq.gz
pbmc_1k_v3_fastqs/pbmc_1k_v3_S1_L002_I1_001.fastq.gz
pbmc_1k_v3_fastqs/pbmc_1k_v3_S1_L001_R1_001.fastq.gz
pbmc_1k_v3_fastqs/pbmc_1k_v3_S1_L002_R1_001.fastq.gz
pbmc_1k_v3_fastqs/pbmc_1k_v3_S1_L002_R2_001.fastq.gz
pbmc_1k_v3_fastqs/pbmc_1k_v3_S1_L001_I1_001.fastq.gz
cellranger 的輸入文件命名規(guī)則為Sample_S1_L00X_R1_001.fastq.gz,以pbmc_1k_v3_S1_L001_R2_001.fastq.gz為例猪叙，樣本為pbmc_1k_v3(v3試劑盒)压恒，樣本在兩條lane上運(yùn)行大州，分別為L(zhǎng)001和L002,該文件為R2文件（文件分為R1,R2和I1）
下載參考基因組
因?yàn)檫@個(gè)例子是人的數(shù)據(jù)，因此需要下載官網(wǎng)上的構(gòu)建好的人的參考基因組索引約為10.6G
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
- 查詢cellranger count的幫助文檔
cellranger count --help
/mnt/home/user.name/yard/apps/cellranger-3.1.0/cellranger-cs/3.1.0/bin
cellranger count (3.1.0)
Copyright (c) 2019 10x Genomics, Inc. All rights reserved.
-------------------------------------------------------------------------------
'cellranger count' quantifies single-cell gene expression.
The commands below should be preceded by 'cellranger':
Usage:
count
--id=ID
[--fastqs=PATH]
[--sample=PREFIX]
--transcriptome=DIR
[options]
count <run_id> [options]
count -h | --help | --version
id:對(duì)你運(yùn)行的項(xiàng)目起個(gè)名字，可以任意取名（輸出結(jié)果在建文件夾時(shí)以這個(gè)名字命名）
fastqs:包含fastq文件的路徑
sample：如果上述路徑中包含的文件不只一個(gè)樣本的肝箱，則需要指定該參數(shù)哄褒，該參數(shù)是根據(jù)fastq文件名的前綴對(duì)文件進(jìn)行識(shí)別的稀蟋，可以用來(lái)區(qū)分不同的樣本
transcriptome：用來(lái)保存參考基因組的路徑
注：換行符 \
- 運(yùn)行cellranger count
cellranger count --id=run_count_1kpbmcs \
--fastqs=/mnt/home/user.name/yard/run_cellranger_count/pbmc_1k_v3_fastqs \
--sample=pbmc_1k_v3 \
--transcriptome=/mnt/home/user.name/yard/run_cellranger_count/refdata-cellranger-GRCh38-3.0.0
正確的輸出為
/mnt/yard/user.name/yard/apps/cellranger-3.1.0/cellranger-cs/3.1.0/bin
cellranger count (3.1.0)
Copyright (c) 2019 10x Genomics, Inc. All rights reserved.
-------------------------------------------------------------------------------
Martian Runtime - '3.1.0-v3.2.3'
...
Pipestance completed successfully!
2019-09-12 15:39:08 Shutting down.
Saving pipestance info to run_count_1kpbmcs/run_count_1kpbmcs.mri.tgz
當(dāng)命令行中出現(xiàn)“==Pipestance completed successfully!==”時(shí)煌张，說(shuō)明運(yùn)行成功
- 對(duì)輸出文件進(jìn)行探索
該實(shí)例中的數(shù)據(jù)文件夾名為run_count_1kpbmcs
其中的文件為
analysis
cloupe.cloupe
filtered_feature_bc_matrix
filtered_feature_bc_matrix.h5
metrics_summary.csv
molecule_info.h5
possorted_genome_bam.bam
possorted_genome_bam.bam.bai
raw_feature_bc_matrix
raw_feature_bc_matrix.h5
web_summary.html
