{"id":27305,"date":"2024-01-31T17:07:28","date_gmt":"2024-01-31T09:07:28","guid":{"rendered":"http:\/\/www.biocloudservice.com\/wordpress\/?p=27305"},"modified":"2024-01-31T17:07:29","modified_gmt":"2024-01-31T09:07:29","slug":"%e8%b6%85%e7%ae%80%e5%8d%95%ef%bc%81%e4%bd%bf%e7%94%a8spaceranger%e5%81%9a%e7%a9%ba%e9%97%b4%e8%bd%ac%e5%bd%95%e7%bb%84%e5%88%86%e6%9e%90","status":"publish","type":"post","link":"http:\/\/www.biocloudservice.com\/wordpress\/?p=27305","title":{"rendered":"\u8d85\u7b80\u5355\uff01\u4f7f\u7528spaceranger\u505a\u7a7a\u95f4\u8f6c\u5f55\u7ec4\u5206\u6790"},"content":{"rendered":"

\u4eca\u5929\u5c0f\u679c\u5e26\u5927\u5bb6\u4f7f\u7528spaceranger\u8fdb\u884c\u7a7a\u95f4\u8f6c\u5f55\u7ec4\u7684\u5206\u6790\u3002<\/p>\n

\u7a7a\u95f4\u8f6c\u5f55\u7ec4\u7b80\u4ecb<\/h2>\n

\u4f20\u7edf\u7684\u5355\u7ec6\u80de\u6d4b\u5e8f\uff0c\u662f\u5c06\u7ec6\u80de\u7ec4\u7ec7\u5236\u5907\u6210\u7ec6\u80de\u60ac\u6d4a\u6db2\uff0c\u8fd9\u6837\u4e00\u6765\u5c31\u4e22\u5931\u4e86\u7ec6\u80de\u6240\u5904\u4f4d\u7f6e\u7684\u7a7a\u95f4\u4fe1\u606f\u3002\u7a7a\u95f4\u8f6c\u5f55\u7ec4\uff08spatial gene expression\uff09, \u7b80\u79f0ST-seq, \u5728\u6b64\u57fa\u7840\u4e4b\u4e0a\u5c06\u8f6c\u5f55\u7ec4\u5b66\uff0c\u5355\u7ec6\u80de\u6d4b\u5e8f\u6280\u672f\u4ee5\u53ca\u7ec4\u7ec7\u5207\u7247\u6280\u672f\u7ed3\u5408\u8d77\u6765\u7684\u6280\u672f\u3002\u800c\u7ec4\u7ec7\u5207\u7247\u5c06\u63d0\u4f9b\u7ec6\u80de\u7684\u7a7a\u95f4\u4fe1\u606f\u3002<\/p>\n

<\/p>\n

\u901a\u8fc7\u8fdb\u884c\u7a7a\u95f4\u8f6c\u5f55\u7ec4\u6d4b\u5e8f\uff0c\u5c31\u80fd\u77e5\u9053\u4e00\u7247\u7ec4\u7ec7\u7684\u7ec6\u80de\u7c7b\u578b\uff0c\u53ca\u5176\u4f4d\u7f6e\uff0c\u8fd9\u5bf9\u7814\u7a76\u80bf\u7624\u7ec4\u7ec7\u4f1a\u6709\u5f88\u5927\u7684\u5e2e\u52a9\u3002<\/p>\n

10\u00d7genomics Visium \u7a7a\u95f4\u8f6c\u5f55\u7ec4\u6d4b\u5e8f\u5e73\u53f0<\/h2>\n

<\/p>\n

10x Visium \u4f7f\u7528\u7279\u5236\u7684\u73bb\u7247\uff0c\u73bb\u7247\u4e0a\u6709\u6355\u83b7\u533a\u57df\uff08capture area\uff09\uff0c\u6355\u83b7\u533a\u57df\u662f\u4e00\u4e2a6.5*6.5mm \u7684\u6b63\u65b9\u5f62\u533a\u57df\uff0c\u6bcf\u4e2a\u6355\u83b7\u533a\u57df\u5305\u542b\u7ea6 5000 \u4e2a spot\uff0c\u6bcf\u4e2a spot \u4e0a\u7ed3\u5408\u6709\u6355\u83b7\u5e8f\u5217\uff0c\u6355\u83b7\u5e8f\u5217\u6709barcode\u5e8f\u5217\u8bb0\u5f55spot \u4f4d\u7f6e\u4fe1\u606f\u548c poly(dT)\u5e8f\u5217\u7ed3\u5408 mRNA\u7684poly(A)\u5c3e\uff0c\u5728\u6355\u83b7mRNA \u7684\u540c\u65f6\u4e5f\u4fdd\u7559\u4e86 mRNA \u7684\u4f4d\u7f6e\u4fe1\u606f\u3002\u8f6c\u5f55\u672c\u7684\u4f4d\u7f6e\u4fe1\u606f\u7ed3\u5408\u67d3\u8272\u62cd\u7167\u7ed3\u679c\u5373\u53ef\u8fd8\u539f\u8f6c\u5f55\u672c\u5728\u7ec4\u7ec7\u7684\u4f4d\u7f6e\u5206\u5e03\u3002<\/p>\n

<\/p>\n

\u76ee\u524d\u80fd\u505a\u7684\u4e3b\u8981\u8fd8\u662f\u4eba\u7c7b\u548c\u8001\u9f20\uff0c\u5176\u4ed6\u7684\u8fd8\u4e0d\u80fd\u505a\u3002<\/p>\n

\u603b\u4f53\u5de5\u4f5c\u6d41\u7a0b<\/h2>\n

<\/p>\n

\u9996\u5148\u9009\u62e9\u7ec4\u7ec7\u4e0e\u5207\u7247\uff0c\u5c06\u5207\u7247\u653e\u7f6e\u5728\u73bb\u7247\u4e0a\uff0c\u5269\u4e0b\u7684\u5efa\u5e93\u6d4b\u5e8f\u5c31\u548c\u4e8c\u4ee3\u6d4b\u5e8f\u4e00\u81f4\u4e86\u3002<\/p>\n

\u65b0\u9c9c\u51b7\u51bb\uff08FF\uff09\u7ec4\u7ec7\u7684\u7a7a\u95f4\u57fa\u56e0\u8868\u8fbe\u662f\u4f7f\u7528spaceranger\u8ba1\u6570\u8f6f\u4ef6\u6765\u786e\u5b9a\u7684\uff0c\u8be5\u8f6f\u4ef6\u4ee5\u89c6\u89c9\u8f7d\u73bb\u7247\u7684\u663e\u5fae\u955c\u56fe\u50cf\uff08TIFF\u6216JPEG\u683c\u5f0f\uff09\u548c\u6837\u672cFASTQ\u6587\u4ef6\u4f5c\u4e3a\u8f93\u5165\u3002\u8f6f\u4ef6\u6267\u884c\u5bf9\u9f50\u3001\u7ec4\u7ec7\u548c\u57fa\u51c6\u68c0\u6d4b\u4ee5\u53ca\u6761\u5f62\u7801\/UMI\u8ba1\u6570\u3002\u8f93\u51fa\u6355\u83b7\u7279\u5f81\u70b9\u77e9\u9635\u3001\u805a\u7c7b\u548c\u5dee\u5f02\u57fa\u56e0\u8868\u8fbe\uff08DGE\uff09\uff0c\u8fd9\u4e9b\u53ef\u4ee5\u901a\u8fc7\u5728Loupe Browser\u4e2d\u8fdb\u884c\u8fdb\u4e00\u6b65\u5206\u6790\u548c\u53ef\u89c6\u5316\u3002<\/p>\n

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

\u4e0b\u8f7dspacerranger\u8f6f\u4ef6<\/h3>\n

curl -o spaceranger-2.1.0.tar.xz “https:\/\/cf.10xgenomics.com\/releases\/spatial-exp\/spaceranger-2.1.0.tar.xz?Expires=1691721798&Key-Pair-Id=APKAI7S6A5RYOXBWRPDA&Signature=nKggndnkZPv4IEuizxHUM5KSXSbtMV7Tk1ICeslkhK09CuFDeOkPBJQcY1yti7s2fMz8i9b2lySWmBeoFdXMS23~ioITj-L88h0ldpTo7G~pqKvMgIssE5b67C4XZ3FprDdceGIaatl4nt~Vrjg~31FZcbG-JxJAQLFf92XLT1tSfSuovhTb5lFkY3oLTfR6Ozvo7Hbx6bibCqSh4MrEefajch4OvdKIlR0l3svzxX0ExETxBiKJw6PBZJu5qYX6vMtDz2anb0-N4-cbhv5Opl3NlT8Xd-7v3mKDCLN37zM-m4vO9EF6YWG2sBljc7ehxsWQnMWw60VhvtbKVb9ULw__”<\/p>\n

<\/p>\n

\u8f93\u51fa\u8f6f\u4ef6\u7684\u5e2e\u52a9\u6587\u6863\uff0c\u5373\u4e3a\u5b89\u88c5\u6210\u529f\u3002<\/p>\n

\u6570\u636e\u96c6\u4fe1\u606f<\/h3>\n

\u5c0f\u679c\u5728\u672c\u6b21\u6559\u7a0b\u4e2d\u4f7f\u7528\u7684\u662f\u5c0f\u9f20\u5927\u8111\u51a0\u72b6\u5207\u7247\u6570\u636e\u96c6<\/p>\n

\u6570\u636e\u96c6\u7684\u4e3b\u8981\u4fe1\u606f\u5305\u62ec\uff1a<\/p>\n

10\u00b5m\u539a\u7684\u7ec4\u7ec7\u5207\u7247<\/p>\n

\u4f7f\u7528Nikon Ti2-E\u663e\u5fae\u955c\u83b7\u53d6\u7684H&E\u56fe\u50cf<\/p>\n

\u6d4b\u5e8f\u6df1\u5ea6\uff1a\u6bcf\u4e2a\u70b9115569\u4e2a\u8bfb\u53d6\u5bf9<\/p>\n

\u6d4b\u5e8f\u8986\u76d6\u8303\u56f4\uff1areads 1-28bp\uff08\u5305\u62ec16bp\u7684\u7a7a\u95f4\u6761\u5f62\u7801\uff0c12bp\u7684UMI\uff09\uff1breads 2-120bp\uff08\u8f6c\u5f55\u672c\uff09\uff1bi7\u6837\u672c\u6307\u6570-10bp\uff1bi5\u6837\u672c\u6307\u6570-10 bp<\/p>\n

Visium\u5e7b\u706f\u7247\uff1aV19L01-041<\/p>\n

\u6355\u83b7\u533a\u57df\uff1aC1<\/p>\n

\u793a\u4f8b\u6570\u636e\u96c6\u5305\u62ec\u5177\u6709\u57fa\u51c6\u5e27\u6e05\u6670\u89c6\u56fe\u7684\u660e\u573a\u56fe\u50cf\uff0c\u4ece\u800c\u80fd\u591f\u4f7f\u7528spaceranger\u81ea\u52a8\u56fe\u50cf\u5904\u7406\u6d41\u6c34\u7ebf\u3002<\/p>\n

\u4e0b\u8f7d\u6570\u636e\u96c6\u4e0e\u53c2\u8003\u5e8f\u5217<\/h3>\n

# Create datasets folder<\/p>\n

mkdir datasets<\/p>\n

# Download FASTQ to datasets folder<\/p>\n

curl https:\/\/s3-us-west-2.amazonaws.com\/10x.files\/samples\/spatial-exp\/1.1.0\/V1_Adult_Mouse_Brain\/V1_Adult_Mouse_Brain_fastqs.tar -o datasets\/V1_Adult_Mouse_Brain_fastqs.tar<\/p>\n

# Download image file to datasets folder<\/p>\n

curl https:\/\/cf.10xgenomics.com\/samples\/spatial-exp\/1.1.0\/V1_Adult_Mouse_Brain\/V1_Adult_Mouse_Brain_image.tif -o datasets\/V1_Adult_Mouse_Brain_image.tif<\/p>\n

# Download mouse reference<\/p>\n

curl -O https:\/\/cf.10xgenomics.com\/supp\/spatial-exp\/refdata-gex-mm10-2020-A.tar.gz<\/a><\/p>\n

\u89e3\u538b\u7f29\u6570\u636e\u96c6\u4e0e\u53c2\u8003\u5e8f\u5217<\/h3>\n

# \u89e3\u538b\u7f29\u6d4b\u5e8f\u6570\u636e<\/p>\n

tar -xvf V1_Adult_Mouse_Brain_fastqs.tar -C .\/ && rm V1_Adult_Mouse_Brain_fastqs.tar<\/p>\n

# Extract mouse reference transcriptome<\/p>\n

tar -xzvf refdata-gex-mm10-2020-A.tar.gz && rm refdata-gex-mm10-2020-A.tar.gz<\/p>\n

<\/p>\n

\u89e3\u538b\u6210\u529f\u540e\u5c06\u4f1a\u5305\u542b\u4ee5\u4e0a\u6587\u4ef6<\/p>\n

spaceranger\u5206\u6790<\/h2>\n

\u73b0\u5728\u6211\u4eec\u6240\u6709\u7684\u8f93\u5165\u6587\u4ef6(\u53c2\u8003\u57fa\u56e0\uff0c\u6d4b\u5e8f\u6587\u4ef6\uff0c\u5207\u7247\u7167\u7247)\u90fd\u51c6\u5907\u597d\u4e86\uff0c\u5c31\u53ef\u4ee5\u4f7f\u7528spaceranger\u6765\u8fdb\u884c\u5206\u6790\u3002<\/p>\n

spaceranger count –id=”V1_Adult_Mouse_Brain” \\<\/p>\n

–description=”Adult Mouse Brain (Coronal)” \\<\/p>\n

–transcriptome=refdata-gex-mm10-2020-A \\<\/p>\n

–fastqs=V1_Adult_Mouse_Brain_fastqs \\<\/p>\n

–image=V1_Adult_Mouse_Brain_image.tif \\<\/p>\n

–slide=V19L01-041 \\<\/p>\n

–area=C1 \\<\/p>\n

–localcores=16 \\<\/p>\n

–localmem=128<\/p>\n

<\/p>\n

spaceranger\u53c2\u6570\u542b\u4e49<\/p>\n

<\/p>\n

\u8fd0\u884c\u597d\u540e\u5c06\u4f1a\u770b\u5230\u4ee5\u4e0a\u8f93\u51fa\u3002<\/p>\n

\u8f93\u51fa\u7684\u6587\u4ef6\u5305\u62ec<\/p>\n

<\/p>\n