cellranger

一、安装

##Cell Ranger - 4.0.0 (July 7, 2020)

cd /data/software/single_cell

1.1 下载

Self-contained, relocatable tar file. Does not require centralized installation. Contains binaries pre-compiled for CentOS/RedHat 6.0+ and Ubuntu 12.04+. Linux 64-bit – 972 MB – md5sum: cc4ed44a57570f6ecb3ea5881e01511d

wget -O cellranger-4.0.0.tar.gz "https://cf.10xgenomics.com/releases/cell-vdj/cellranger-4.0.0.tar.gz?Expires=1600351663&Policy=eyJTdGF0ZW1lbnQiOlt7IlJlc291cmNlIjoiaHR0cHM6Ly9jZi4xMHhnZW5vbWljcy5jb20vcmVsZWFzZXMvY2VsbC12ZGovY2VsbHJhbmdlci00LjAuMC50YXIuZ3oiLCJDb25kaXRpb24iOnsiRGF0ZUxlc3NUaGFuIjp7IkFXUzpFcG9jaFRpbWUiOjE2MDAzNTE2NjN9fX1dfQ__&Signature=nFPhAQ936rBw6Sn~ERfxff5CEtgk~F9IISOzNUjvjWiLbrpooHcUOk4q1xINBSU4DJILMa2KckRnvR5V7nnoOw4MSOF8YqLsuOqB7D0HBmvF5V9WYa6bs1CC86Muh70B~2VEvV6bravMXdgRE5ah0B-E4P9ZNmf~arxTmXigLH~CJOZ9Uof1PmMJmnxJWWAKnQDDEBrAtQMQkrFPE0cjZ0DI~uWkcq0sNicoz9rBEno0cmiz8XgU0Kz61lVvScWu4SHrRjie4PuoOMxn7yaARliaESGcIzfiayEcNtZi8BYCJK8LKlRX~ypzaHL0a50ZC5KboMw8wccFs-rFyLYiRQ__&Key-Pair-Id=APKAI7S6A5RYOXBWRPDA"

#GRCh38 Reference - 4.0.0 (July 7, 2020) Human reference dataset (GRCh38/Ensembl/10x) required for Cell Ranger V(D)J. Linux 64-bit – 3.3 MB – md5sum: 67e4fff7e80d7f7c87ec16475859c080

wget -c https://cf.10xgenomics.com/supp/cell-vdj/refdata-cellranger-vdj-GRCh38-alts-ensembl-4.0.0.tar.gz

#GRCm38 Reference - 4.0.0 (July 7, 2020)

Mouse reference dataset (GRCm38/Ensembl/10x) required for Cell Ranger V(D)J.
Linux 64-bit – 3.5 MB – md5sum: 1f2e9adbff3c8a01559ccf7310a9d1e7

wget -c https://cf.10xgenomics.com/supp/cell-vdj/refdata-cellranger-vdj-GRCm38-alts-ensembl-4.0.0.tar.gz

1.2解压缩

tar -xzvf cellranger-4.0.0.tar.gz
tar -xzvf refdata-cellranger-vdj-GRCh38-alts-ensembl-4.0.0.tar.gz
tar -xzvf refdata-cellranger-vdj-GRCm38-alts-ensembl-4.0.0.tar.gz

export PATH=/data/software/single_cell/cellranger-4.0.0:$PATH

1.3 测试

cd /data/software/single_cell/test

source /opt/fastone/softwares/hpc-kits/hpc-kits.sh

export PATH=/opt/cellranger-4.0.0:$PATH

cellranger testrun --id=tiny

Running Cell Ranger in test mode...

Martian Runtime - v4.0.0

...

Pipestance completed successfully!

yyyy-mm-dd hh:mm:ss Shutting down.
Saving pipestance info to "tiny/tiny.mri.tgz"

当然,测试结果可以直接发送给cellranger的开发

1.3 VCDJ 可视化

cellranger vdj管道将为单细胞V(D)J实验生成.vloupe文件。 .vloupe文件将在完成的cellranger vdj运行的outs文件夹中找到。

Loupe V(D)J Browser for Windows - 3.0.0 (November 19, 2018) Windows 7 (64-bit) or later 2GB RAM Download – 56 MB – md5sum: bfbc86a81848ea44dfae61e6c9bbfeb0

下载地址:

https://cf.10xgenomics.com/releases/cell-vdj/Loupe-VDJ-Browser-3.0.0.exe?Expires=1601330276&Policy=eyJTdGF0ZW1lbnQiOlt7IlJlc291cmNlIjoiaHR0cHM6Ly9jZi4xMHhnZW5vbWljcy5jb20vcmVsZWFzZXMvY2VsbC12ZGovTG91cGUtVkRKLUJyb3dzZXItMy4wLjAuZXhlIiwiQ29uZGl0aW9uIjp7IkRhdGVMZXNzVGhhbiI6eyJBV1M6RXBvY2hUaW1lIjoxNjAxMzMwMjc2fX19XX0_&Signature=oA7~CvZitD60paVH3gUySwUW7OenByfCBiG8pvfKwdn9w8rQYbGq0SH8Ygc9mM~I5oDjUe9QZ-YDh8y~5rU5W5b83lDznkjBCnmvTLSTOKnn7okiOL~K2bGWe8494uGytgGza1fccfElNCR5eDCTMf--VDpvd7pbVEoKot5MEKuQMmRq-DjNwq0EhtZOMngV9WL3cXskenVNpgk7t2o-wVEIb6FCPC0Odz8Yup-lHdbLBVGghYGhxuN1Wwr843w~cd1hHZb2n1e4dPV4JZNNvGmjLc4Urs6IvrgDdTYoa-Mmnz0tbumXCjcHKgeVUnzv04GAb35rI14VA4RM6Yokqg__&Key-Pair-Id=APKAI7S6A5RYOXBWRPDA

二、分析流程

cd /data/user/sam/project/scBCR/1.analysis/input/X101SC20090516-Z01-J001

1.质控 150m,g01

source /data/user/sam/project/meta/miniconda2/bin/activate root
conda activate metawrap-env
export PERL5LIB=/data/user/sam/project/meta/miniconda2/envs/metawrap-env/lib/5.26.2/

time metawrap read_qc -1 Rawdata/G_MB_1/G_MB_1_1.fastq.gz -2 Rawdata/G_MB_1/G_MB_1_2.fastq.gz -t 60 -o 1.clean_data/G_MB_1 --skip-bmtagger

cd /data/user/sam/project/scBCR/1.analysis/input/X101SC20090516-Z01-J001/1.clean_data/G_MB_1

mv G_MB_1_1_val_1.fq.gz G_MB_1_S1_L001_R1_001.fastq.gz
mv G_MB_1_2_val_2.fq.gz G_MB_1_S1_L001_R2_001.fastq.gz

conda deactivate

2. vdj聚类(4个小时,50核)

/data/software/single_cell/cellranger-4.0.0/bin/cellranger --help

time /data/software/single_cell/cellranger-4.0.0/bin/cellranger vdj --reference  /data/software/single_cell/refdata-cellranger-vdj-GRCm38-alts-ensembl-4.0.0 --fastqs //data/user/sam/project/scBCR/1.analysis/input/X101SC20090516-Z01-J001/1.clean_data/G_MB_1  --id cellranger  --localcores=50  --sample G_MB_1

参考资料

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