单细胞ATAC实战01: CellRanger

2023-03-28 19:09| 来源: 网络整理| 查看: 265

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原理

It enables profiling of the open chromatin landscape at single cell resolution.

安装cellranger-atac

https://support.10xgenomics.com/single-cell-atac/software/downloads/latest?

mkdir -p APP/bin cd ~/APP curl -o cellranger-atac-2.1.0.tar.gz "https://cf.10xgenomics.com/releases/cell-atac/cellranger-atac-2.1.0.tar.gz?Expires=1679232379&Policy=eyJTdGF0ZW1lbnQiOlt7IlJlc291cmNlIjoiaHR0cHM6Ly9jZi4xMHhnZW5vbWljcy5jb20vcmVsZWFzZXMvY2VsbC1hdGFjL2NlbGxyYW5nZXItYXRhYy0yLjEuMC50YXIuZ3oiLCJDb25kaXRpb24iOnsiRGF0ZUxlc3NUaGFuIjp7IkFXUzpFcG9jaFRpbWUiOjE2NzkyMzIzNzl9fX1dfQ__&Signature=Nsc4TJ5H0XyVlPDTmVZjZGnWO0rfU7TNB~SM7f4JbP2pGgOp~vWcLj1wPA6o8w~Pvm3yOO8eqiA5s72PpdeR~WG1KEIS6Rx9aYR-0GkQLlGfxLXG1aJ3SdXg8PJ8jz2cqS2DUMS4Jrh2sNIDp7r7rPcf3Znq34HP9d-x-6W8Ct53ybSNuvz12EMeeJemCEldFKYSUy1Q6GPPp8Ufg5CQLzfXKx4DQ5DqMBQD-aaKJ6PAGx6jFTOGwio76i~24AQEzR2fNJgOtKSI6gyA3I9c-2oyrzMrt0J3zakhOzyej0M8~zmXp5gbtxV6GbWioZj5MwRvLKWLHmkkDQJqczzxjg__&Key-Pair-Id=APKAI7S6A5RYOXBWRPDA"# 软连接 ~/APP/bin已加入path md5sum *gz tar -xf *gz rm -f *gz ln -s ~/APP/cellranger-atac-2.1.0/bin/cellranger-atac ~/APP/bin/cellranger-atac export PATH=$HOME/APP/bin:$PATH # 测试 nohup cellranger-atac testrun --id=tiny &> testrun.txt &下载fastq数据

下载10X官网提供的10K和5K PBMC数据集

https://www.10xgenomics.com/resources/datasets/5-k-peripheral-blood-mononuclear-cells-pbm-cs-from-a-healthy-donor-next-gem-v-1-1-1-1-standard-2-0-0

https://www.10xgenomics.com/resources/datasets/10-k-peripheral-blood-mononuclear-cells-pbm-cs-from-a-healthy-donor-next-gem-v-1-1-1-1-standard-2-0-0

cd ~/Project/ATAC mkdir fastqs quantify cd fastqs curl -O https://s3-us-west-2.amazonaws.com/10x.files/samples/cell-atac/2.0.0/atac_pbmc_10k_nextgem/atac_pbmc_10k_nextgem_fastqs.tar curl -O https://cf.10xgenomics.com/samples/cell-atac/2.0.0/atac_pbmc_5k_nextgem/atac_pbmc_5k_nextgem_fastqs.tar tar -xf *tar rm -f *tar cd atac_pbmc_10k_nextgem_fastqs mv * .. cd .. cd atac_pbmc_5k_nextgem_fastqs mv * .. cd .. rm -rf atac_pbmc_10k_nextgem_fastqs/ atac_pbmc_5k_nextgem_fastqs/

Sequenced on Illumina NovaSeq with approximately 39/41k read pairs per cell

50 bp Read 18 bp i7 index (sample index)16 bp i5 (10x Barcode)49 bp Read 21.4G atac_pbmc_10k_nextgem_S1_L001_I1_001.fastq.gz 5.7G atac_pbmc_10k_nextgem_S1_L001_R1_001.fastq.gz 3.2G atac_pbmc_10k_nextgem_S1_L001_R2_001.fastq.gz 5.7G atac_pbmc_10k_nextgem_S1_L001_R3_001.fastq.gz 1.4G atac_pbmc_10k_nextgem_S1_L002_I1_001.fastq.gz 5.7G atac_pbmc_10k_nextgem_S1_L002_R1_001.fastq.gz 3.2G atac_pbmc_10k_nextgem_S1_L002_R2_001.fastq.gz 5.7G atac_pbmc_10k_nextgem_S1_L002_R3_001.fastq.gz 787M atac_pbmc_5k_nextgem_S1_L001_I1_001.fastq.gz 3.2G atac_pbmc_5k_nextgem_S1_L001_R1_001.fastq.gz 1.8G atac_pbmc_5k_nextgem_S1_L001_R2_001.fastq.gz 3.1G atac_pbmc_5k_nextgem_S1_L001_R3_001.fastq.gz 788M atac_pbmc_5k_nextgem_S1_L002_I1_001.fastq.gz 3.2G atac_pbmc_5k_nextgem_S1_L002_R1_001.fastq.gz 1.8G atac_pbmc_5k_nextgem_S1_L002_R2_001.fastq.gz 3.1G atac_pbmc_5k_nextgem_S1_L002_R3_001.fastq.gzReference

10X官网提供了人和小鼠的参考索引，其他物种可以参考官网代码自行构建

https://support.10xgenomics.com/single-cell-atac/software/release-notes/references

https://support.10xgenomics.com/single-cell-atac/software/downloads/latest?

cd ~/DataHub/Genomics/10X # Human ATAC reference (GRCh38) md5sum:2f12f6016197869e21e5559827002716 curl -O https://cf.10xgenomics.com/supp/cell-atac/refdata-cellranger-arc-GRCh38-2020-A-2.0.0.tar.gz # Mouse ATAC reference md5sum: a2c1cc9b8dff2a2ef36971d7c31c8304 curl -O https://cf.10xgenomics.com/supp/cell-atac/refdata-cellranger-arc-mm10-2020-A-2.0.0.tar.gz md5sum *gz tar -xf *gz rm -f *gzcount#>>>quantify.sh>>> human_index_dir=~/DataHub/Genomics/10X/refdata-cellranger-arc-GRCh38-2020-A-2.0.0 mouse_index_dir=~/DataHub/Genomics/10X/refdata-cellranger-arc-mm10-2020-A-2.0.0 fastqs_dir=~/Project/ATAC/fastqs output_dir=~/Project/ATAC/quantify cd ${output_dir} ls ${fastqs_dir} | cut -d '_' -f 1-4 | uniq | while read i do cellranger-atac count \ --id $i \ --reference ${human_index_dir} \ --fastqs ${fastqs_dir} \ --sample $i \ --localcores 12 \ --localmem 128 done #

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