{"id":26023,"date":"2024-01-24T17:08:31","date_gmt":"2024-01-24T09:08:31","guid":{"rendered":"http:\/\/www.biocloudservice.com\/wordpress\/?p=26023"},"modified":"2024-01-24T17:08:33","modified_gmt":"2024-01-24T09:08:33","slug":"10xgenomics%e5%8d%95%e7%bb%86%e8%83%9e%e6%b5%8b%e5%ba%8f%e5%88%86%e6%9e%90%ef%bc%88%e4%b8%80%ef%bc%89%e7%8e%af%e5%a2%83%e6%90%ad%e5%bb%ba%e4%b8%8ecell-ranger%e6%95%b0%e6%8d%ae%e5%a4%84%e7%90%86","status":"publish","type":"post","link":"http:\/\/www.biocloudservice.com\/wordpress\/?p=26023","title":{"rendered":"10\u00d7genomics\u5355\u7ec6\u80de\u6d4b\u5e8f\u5206\u6790\uff08\u4e00\uff09\u73af\u5883\u642d\u5efa\u4e0eCell Ranger\u6570\u636e\u5904\u7406"},"content":{"rendered":"

10\u00d7genomics\u56e0\u5176\u5206\u9009\u7ec6\u80de\u6548\u679c\u597d\uff0c\u901a\u91cf\u9ad8\u7b49\u4f18\u70b9\u5df2\u6210\u4e3a\u4e3b\u6d41\u7684\u5355\u7ec6\u80de\u6d4b\u5e8f\u5e73\u53f0\u3002<\/p>\n

\u5c0f\u679c\u4e0a\u671f\u5bf9\u5b83\u7684\u539f\u7406\u8fdb\u884c\u4e86\u4ecb\u7ecd\uff0c\u611f\u5174\u8da3\u7684\u5c0f\u4f19\u4f34\u53ef\u4ee5\u53bb\u770b\u3002\u8fd9\u671f\u5c0f\u679c\u5c06\u4ecb\u7ecd10\u00d7genomics\u5355\u7ec6\u80de\u6d4b\u5e8f\u5206\u6790\u73af\u5883\u7684\u642d\u5efa\u4e0e\u752810\u00d7\u5e73\u53f0\u5b98\u65b9\u51fa\u7684\u6570\u636e\u5904\u7406\u6d41\u7a0bCell Ranger\u8fdb\u884c\u6570\u636e\u5904\u7406\u4e0ecount\u8868\u8fbe\u77e9\u9635\u7684\u751f\u6210\u3002<\/p>\n

<\/p>\n

\u901a\u5e38\u8fd9\u662f\u5355\u7ec6\u80de\u5206\u6790\u7684\u6574\u4e2a\u6d41\u7a0b\u3002<\/p>\n

\u4e0e\u8bb8\u591a\u7ec4\u5b66\u5206\u6790\u4e00\u6837\uff0c\u5148\u5c06FASTQ\u6587\u4ef6\u6bd4\u5bf9\uff0c\u8f6c\u6362\u4e3a\u4e8c\u8fdb\u5236\u7684BAM\u6587\u4ef6\uff0c\u7136\u540e\u8fdb\u884c\u57fa\u56e0\u8868\u8fbe\u8ba1\u6570\u3002\u62ff\u5230\u8868\u8fbe\u77e9\u9635\u540e\u624d\u80fd\u8fdb\u884c\u4e0b\u4e00\u6b65\u7684\u5206\u6790\u3002<\/p>\n

\u4eca\u5929\u5c0f\u679c\u5e26\u5927\u5bb6\u5b8c\u6210\u6d41\u7a0b\u7684\u642d\u5efa\u4e0e\u4e0a\u6e38\u7684\u7684\u6570\u636e\u5904\u7406\u3002\u5c31\u662f\u8fd9\u5f20\u56fe\u4e0a\u9762\u4e09\u4e2a\u6b65\u9aa4\u3002<\/p>\n

\u4e00\u3001\u51c6\u5907\u5de5\u4f5c<\/h2>\n

\uff08\u4e00\uff09\u8f6f\u4ef6\u5b89\u88c5<\/h3>\n

1.CellRanger<\/h4>\n

Cell Ranger\u662f10\u00d7genomics\u5b98\u65b9\u51fa\u7684\u6570\u636e\u5904\u7406\u5206\u6790\u5de5\u5177\uff0c\u5b83\u7684\u9002\u914d\u5ea6\u597d\uff0c\u529f\u80fd\u5f3a\u5927\uff0c\u64cd\u4f5c\u7b80\u5355\u7b49\u4f18\u70b9\uff0c\u6210\u4e3a\u4e86\u5355\u7ec6\u80de\u6d4b\u5e8f\u6570\u636e\u5206\u6790\u7684\u5fc5\u5907\u5de5\u5177\u3002<\/p>\n

\u5728\u83b7\u5f97\u5355\u7ec6\u80de\u7684\u6d4b\u5e8f\u6570\u636e\u540e\uff0c\u4f7f\u7528Cell Ranger\u6765\u8fdb\u884c\u6570\u636e\u7684\u5904\u7406\u4e0e\u5206\u6790\u7684\u5de5\u4f5c\u6d41\u7a0b\u3002<\/p>\n

What is Cell Ranger? -Software -Single Cell Gene Expression -Official 10x Genomics Support<\/a><\/p>\n

\u4e0a\u9762\u662f10\u00d7genomics\u6d4b\u5e8f\u5e73\u53f0Cell Ranger\u4e3b\u9875\u94fe\u63a5\uff0c\u8be6\u7ec6\u4ecb\u7ecd\u4e86Cell Ranger\u662f\u4ec0\u4e48\u4e0e\u600e\u4e48\u7528\u3002<\/p>\n

<\/p>\n

\u4e0b\u8f7d\u9700\u8981\u586b\u5199\u4fe1\u606f\u5b8c\u6210\u6ce8\u518c\u3002<\/p>\n

<\/p>\n

\u590d\u5236\u4e0b\u8f7d\u94fe\u63a5\u5230linux\u547d\u4ee4\u884c\uff0c\u8fd0\u884c\u5e76\u4e0b\u8f7d<\/p>\n

<\/p>\n

#\u968f\u540e\u89e3\u538b\u7f29\u4e0b\u8f7d\u7684\u8f6f\u4ef6\u5305<\/p>\n

tar -zxvf cellranger-7.1.0.tar.gz<\/p>\n

<\/p>\n

\u5728\u547d\u4ee4\u884c\u6572\u4e00\u4e0b\uff0cok\uff0c\u8f6f\u4ef6\u5b89\u88c5\u597d\u4e86\u3002<\/p>\n

Loupe Browser<\/h4>\n

Loupe Browser\u662f\u4e00\u4e2a\u684c\u9762\u5e94\u7528\u7a0b\u5e8f\uff0c\u63d0\u4f9b\u4ea4\u4e92\u5f0f\u53ef\u89c6\u5316\u5206\u6790\u6765\u81ea\u4e0d\u540c 10x \u57fa\u56e0\u7ec4\u5b66\u89e3\u51b3\u65b9\u6848\u7684\u6570\u636e\u7684\u529f\u80fd\u3002\u5b83\u80fd\u8f7b\u677e\u67e5\u8be2 10x \u57fa\u56e0\u7ec4\u5b66\u6570\u636e\u7684\u4e0d\u540c\u89c6\u56fe\u5feb\u901f\u6df1\u5165\u4e86\u89e3\u6570\u636e\u3002 <\/p>\n

<\/p>\n

\u4e0b\u8f7d\u4ecd\u7136\u9700\u8981\u586b\u5199\u4e00\u6b21\u4fe1\u606f\u3002\u586b\u5199\u597d\u4fe1\u606f\u540e\u4fbf\u53ef\u4ee5\u4e0b\u8f7d\u3002<\/p>\n

\u5728windows\u7cfb\u7edf\u4e0b\u6253\u5f00\u5b89\u88c5\u5305\uff0c\u8fdb\u884c\u5b89\u88c5\u5373\u53ef\u3002<\/p>\n

<\/p>\n

\u8fd9\u662f\u5e94\u7528\u7684\u754c\u9762<\/p>\n

3.bcl2fastq<\/h4>\n

\u662fillumina\u5e73\u53f0\u5185\u7f6e\u7684\u4e00\u4e2a\u8f6f\u4ef6\uff0cCell Ranger\u6ca1\u6709\u96c6\u6210\u8fd9\u4e2a\u8f6f\u4ef6\uff0c\u6211\u4eec\u9700\u8981\u81ea\u5df1\u53bb\u4e0b\u8f7d\u3002\u6709\u4e86\u8fd9\u4e2a\u8f6f\u4ef6\u4e4b\u540e\uff0cCell Ranger\u53ef\u4ee5\u5b8c\u6210\u4ecebcl\u6d4b\u5e8f\u6587\u4ef6\uff0c\u5230\u6700\u540e\u7ed3\u679c\u751f\u6210\uff0c\u4e00\u6761\u9f99\u5f0f\u5730\u5b8c\u6210\u3002<\/p>\n

<\/p>\n

bcl2fastq and bcl2fastq2 Conversion Software Downloads (illumina.com.cn)<\/a><\/p>\n

bcl2fastq\u7684\u7f51\u7ad9<\/p>\n

\u540c\u6837\u9700\u8981\u6ce8\u518c\u586b\u5199\u4fe1\u606f\uff0c\u5c31\u53ef\u4ee5\u8fdb\u4e00\u6b65\u5b89\u88c5\u4e86<\/p>\n

https:\/\/support.illumina.com.cn\/content\/dam\/illumina-support\/documents\/downloads\/software\/bcl2fastq\/bcl2fastq2-v2-18-0-12-tar.zip<\/p>\n

\u8fd9\u91cc\u8d34\u4e0a\u4e0b\u8f7d\u7684\u5730\u5740<\/p>\n

#\u89e3\u538b\u7f29<\/p>\n

unzip bcl2fastq\/bcl2fastq2-v2-18-0-12-tar.zip<\/p>\n

tar -zxvf bcl2fastq\/bcl2fastq2-v2-18-0-12-tar.gz<\/p>\n

#\u7f16\u8bd1\u8f6f\u4ef6<\/p>\n

cd bcl2fastq2<\/p>\n

chmod ugo+x src\/configure<\/p>\n

chmod ugo+x src\/cmake\/bootstrap\/installCmake.sh<\/p>\n

mkdir bin<\/p>\n

cd bin<\/p>\n

..\/src\/configure –prefix=${PWD}\/bin<\/p>\n

make<\/p>\n

make install<\/p>\n

4.\u521b\u5efaconda\u73af\u5883\u5b89\u88c5\u8f6f\u4ef6<\/h4>\n

#\u5b89\u88c5mamba<\/p>\n

conda install -y mamba<\/p>\n

#\u521b\u5efa\u865a\u62df\u73af\u5883<\/p>\n

conda crate -n -y singlecell<\/p>\n

#\u6fc0\u6d3b\u73af\u5883<\/p>\n

conda activate singlecell<\/p>\n

#\u4e0b\u8f7d\u8f6f\u4ef6<\/p>\n

mamba install -y fastqc<\/p>\n

mamba install -u multiqc<\/p>\n

mamba install -y fastp<\/p>\n

mamba install -y star<\/p>\n

mamba install -y bioconductor-singler<\/p>\n

mamba install -y samtools<\/p>\n

mamba install -y featurecounts<\/p>\n

mamba install -y r-seurat<\/p>\n

mamba install -y umi_tools<\/p>\n

\u6570\u636e\u4e0b\u8f7d<\/h3>\n

\u53c2\u8003\u5e8f\u5217\u4e0b\u8f7d<\/h4>\n

#\u4eba\u57fa\u56e0\u7ec4<\/p>\n

wget https:\/\/cf.10xgenomics.com\/supp\/cell-exp\/refdata-gex-GRCh38-2020-A.tar.gz<\/p>\n

#\u5c0f\u9f20\u57fa\u56e0\u7ec4<\/p>\n

wget https:\/\/cf.10xgenomics.com\/supp\/cell-exp\/refdata-gex-mm10-2020-A.tar.gz<\/p>\n

#\u4eba\u548c\u5c0f\u9f20\u6df7\u5408<\/p>\n

wget https:\/\/cf.10xgenomics.com\/supp\/cell-exp\/refdata-gex-GRCh38-and-mm10-2020-A.tar.gz<\/p>\n

#Sample Index Set Sequences<\/p>\n

wget https:\/\/cf.10xgenomics.com\/supp\/cell-exp\/chromium-shared-sample-indexes-plate.csv<\/p>\n

wget https:\/\/cf.10xgenomics.com\/supp\/cell-exp\/chromium-single-cell-sample-indexes-plate-v1.csv<\/p>\n

wget https:\/\/cf.10xgenomics.com\/supp\/cell-exp\/gemcode-single-cell-sample-indexes-plate.csv<\/a><\/p>\n

2.\u7ec3\u4e60\u6570\u636e\u4e0b\u8f7d<\/h4>\n

#pbmc 1k\u6570\u636e<\/p>\n

wget https:\/\/cf.10xgenomics.com\/samples\/cell-exp\/3.0.0\/pbmc_1k_v3\/pbmc_1k_v3_fastqs.tar<\/p>\n

1000\u4e2a\u5355\u7ec6\u80de\u7684\u6d4b\u5e8f\u6570\u636e<\/p>\n

\u4f7f\u7528cellranger\u5206\u6790\u5355\u7ec6\u80de\u6570\u636e<\/h2>\n

Basecalling<\/h3>\n

1.\u5c06\u6d4b\u5e8f\u539f\u59cb\u6570\u636ebcl\u6587\u4ef6\u8f6c\u6362\u4e3afastq\u6587\u4ef6<\/h4>\n

#\u6848\u4f8b\u6570\u636e\uff0cilluminca\u6d4b\u5e8f\u539f\u59cb\u6570\u636e<\/p>\n

wget https:\/\/cf.10xgenomics.com\/supp\/cell-exp\/cellranger-tiny-bcl-1.2.0.tar.gz<\/p>\n

wget https:\/\/cf.10xgenomics.com\/supp\/cell-exp\/cellranger-tiny-bcl-simple-1.2.0.csv<\/p>\n

tar -zxvf cellranger-tiny-bcl-1.2.0.tar.gz<\/p>\n

<\/p>\n

\u8fd9\u662f\u8fd9\u4e2a\u539f\u59cb\u6d4b\u5e8f\u6570\u636e\u6240\u5305\u542b\u7684\u5185\u5bb9\uff0c\u6570\u636e\u5728\u8fd9\u4e2aData\u76ee\u5f55\u4e0b\u9762\u3002<\/p>\n

<\/p>\n

simple\u6587\u4ef6\u662f\u4e2a\u8868\u683c\u3002\u8fd9\u4e2a\u6848\u4f8b\u6570\u636e\u53ea\u6709\u4e00\u4e2a\u6837\u672c\u30021\u4ee3\u8868\u7b2c\u4e00\u4e2a\u6837\u672c\uff0c\u7136\u540e\u6837\u672c\u540d\uff0c\u6700\u540e\u662fIndex\u53f7\u7801\u3002<\/p>\n

cellranger mkfastq –id=output –run=cellranger-tiny-bcl-1.2.0 –csv=cellranger-tiny-bcl-simple-1.2.0.csv –localcores=12 –localmem=32<\/p>\n

\u4f7f\u7528cellranger mkfastq\u8f6f\u4ef6<\/p>\n

–id: \u8f93\u5165\u7684\u6587\u4ef6\u5939<\/p>\n

–run: \u8f93\u5165\u7684\u6587\u4ef6<\/p>\n

–csv: \u6570\u636e\u7684\u5217\u8868<\/p>\n

–localcores: \u63a7\u5236CPU\u6838\u5fc3\u6570<\/p>\n

–localmem: \u63a7\u5236\u5185\u5b58\u6570\u76ee<\/p>\n

<\/p>\n

\u975e\u5e38\u7b80\u5355\uff0c\u53ea\u8981\u8f93\u5165\u6587\u4ef6\uff0c\u53ca\u8f93\u51fa\u7684\u6587\u4ef6\u5939\u5c31\u884c\u4e86\u3002\u56e0\u4e3a\u6570\u636e\u5f88\u5c0f\uff0c\u5f88\u5feb\u5c31\u8fd0\u884c\u597d\u4e86\u3002<\/p>\n

<\/p>\n

\u751f\u6210\u7684fastq\u5c31\u5728\u8fd9\u4e2a\u6587\u4ef6\u5939\u91cc\u3002<\/p>\n

<\/p>\n

Test_sample\u5c31\u662f\u6700\u7ec8\u7684\u7ed3\u679c\u3002\u6587\u4ef6\u540d\u4f1a\u6839\u636esimple\u6587\u4ef6\u7684\u540d\u79f0\u547d\u540d\u3002<\/p>\n

<\/p>\n

\u6253\u5f00\u7b2c\u4e00\u4e2a\u770b\u4e00\u4e0b\uff0c\u5176\u4e3aIndex\u5e8f\u5217\u3002<\/p>\n

<\/p>\n

Read1\u4e3abarcod+UMI<\/p>\n

<\/p>\n

Read2\u662f\u771f\u6b63\u7684\u6211\u4eec\u60f3\u8981\u7684\u6d4b\u5e8f\u5e8f\u5217\u3002<\/p>\n

\u8be5\u90e8\u5206\u4e3a\u6f14\u793a\u5982\u4f55\u5c06bcl\u8f6c\u5316\u4e3afastq\u3002<\/p>\n

\u8d28\u63a7<\/h4>\n

\u63a5\u4e0b\u6765\u6211\u4eec\u5bf9\u7ec3\u4e60\u6570\u636e\u8fdb\u884c\u5904\u7406\uff0c\u8fd9\u91cc\u6211\u4eec\u6253\u5f00\u6848\u4f8b\u7684\u6570\u636e\u3002\u4f7f\u7528\u901a\u914d\u7b26*\u5339\u914d\u6240\u6709fastq\u7684read2\u3002\u7136\u540e\u8fdb\u884c\u8d28\u63a7\u3002<\/p>\n

#\u5bf9read2 \u8fdb\u884c\u8d28\u63a7<\/p>\n

ll pbmc_1k_v3_fastqs\/pbmc_1k_v3*_R2_001.fastq.gz<\/p>\n

fastqc -t 12 -f fastq -o qc1 pbmc_1k_v3_fastqs\/pbmc_1k_v3*_R2_001.fastq.gz<\/p>\n

<\/p>\n

<\/p>\n

\u8d28\u63a7\u7ed3\u679c\uff0c\u663e\u793a\u6d4b\u5e8f\u6570\u636e\u8d28\u91cf\u8f83\u597d\u3002<\/p>\n

10\u00d7genomics\u7684\u6d4b\u5e8f\u6570\u636e\u901a\u5e38\u8d28\u91cf\u8f83\u597d\uff0c\u6240\u4ee5\u7edd\u5927\u90e8\u5206\u4e0d\u7528\u8fdb\u884c\u8d28\u63a7\u4ee5\u53ca\u8fc7\u6ee4\u3002<\/p>\n

\u5b9e\u5728\u8fc7\u6ee4\u7684\u8bdd\u5462\uff0c\u7531\u4e8eread1\u548cread2\u4e0d\u4e00\u6837\uff0c\u524d\u8005\u662fbarcode\uff0c\u540e\u8005\u662f\u6d4b\u5e8f\u6570\u636e\uff0c\u5bf9\u6d4b\u5e8f\u6570\u636e\u8fc7\u6ee4\u6389\u4ee5\u540e\uff0c\u4e8c\u8005\u7d22\u5f15\u4e0d\u5339\u914d\uff0c\u5f15\u8d77\u64cd\u4f5c\u9ebb\u70e6\u3002<\/p>\n

\u751f\u6210\u8868\u8fbe\u77e9\u9635<\/h3>\n

<\/p>\n

Cellranger count\u7684\u9009\u9879\u53c2\u6570\uff0c\u8be5\u8f6f\u4ef6\u4e3b\u6253\u7684\u5c31\u662f\u4e00\u4e2a\u7b80\u5355\u65b9\u4fbf\uff0c\u7528\u8d77\u6765\u7b80\u5355\u7edd\u5bf9\u662f\u4e2a\u4f18\u70b9\u3002<\/p>\n

\u8f93\u5165\u524d\u9762\u4e09\u4e2a\u53c2\u6570\u5c31\u53ef\u4ee5\u5de5\u4f5c\u4e86\u3002<\/p>\n

#\u8ba1\u7b97count \u77e9\u9635\uff0cpbmc 1000\u4e2a\u7ec6\u80de\uff0c\u7b49\u53f7\u4e4b\u95f4\u4e0d\u8981\u6709\u7a7a\u683c<\/p>\n

cellranger count –id=run_count_1kpbmcs –fastqs=pbmc_1k_v3_fastqs –sample=pbmc_1k_v3 \\<\/p>\n

–transcriptome=refdata-gex-GRCh38-2020-A\/ \\<\/p>\n

–localcores=12 –localmem=32<\/p>\n

\u8bbe\u7f6e\u8f93\u5165\u8f93\u51fa\uff0c\u5269\u4e0b\u7684\u6309\u7167\u9ed8\u8ba4\u7684\u5c31\u884c\u4e86\u3002<\/p>\n

<\/p>\n

\u8fd9\u6837\u5b83\u5c31\u8fd0\u884c\u8d77\u6765\u4e86\u3002<\/p>\n

<\/p>\n

\u6700\u7ec8\u7684\u7ed3\u679c\u5728outs\u91cc\u9762<\/p>\n

<\/p>\n

\u5c0f\u679c\u4eca\u5929\u7684\u8bb2\u89e3\u5c31\u5230\u8fd9\u91cc\u4e86~\u4e0b\u671f\u6211\u5c06\u4f1a\u5e26\u6765\u5bf9\u5355\u7ec6\u80de\u6d4b\u5e8f\u5206\u6790\u7ed3\u679c\u7684\u89e3\u8bfb~\u6211\u4eec\u4e0d\u89c1\u4e0d\u6563<\/p>\n

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