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10X Visium HD空转流程1·Space Ranger v4.0.1的使用

news 2025/8/21 7:41:20

1. 下载并解压Space Ranger v4.0.1

curl -o spaceranger-4.0.1.tar.gz "https://cf.10xgenomics.com/releases/spatial-exp/spaceranger-4.0.1.tar.gz?Expires=1751416655&Key-Pair-Id=APKAI7S6A5RYOXBWRPDA&Signature=VXCX5mVKYbxc7g-0bXPhLdERaMEM5nygTPbRUTGppwHFiNoq3z-EgYs5codalwb0f6wbo3IFsM9GhHDwsg0VXes80erdeZi77bJ8p83WHX7LawneT9d3iMVUB5yy7JbMkl9y-Km~yGQnE2ol46WLUWYMLbErhQygfNN4VMQi5xgfc1I1XJcDzEfi-tNEesD3-yXDUL8Gb7uBWuhS25ODgCxF0J97Z4oyNcYofyUu~hAGBRHOu9YRXyCDfHfA7MGLXC5gPRgX3kaOb3FGq3YYTTiio-d5IMkQ64m0rfWkxDINMVdFGpp7uck59bIZ0CmN5SRH5415NkaFY6Rz2oD4FQ__"
tar -xzvf spaceranger-4.0.1.tar.gz

2. 设置环境变量

echo 'export PATH=$PATH:/project/Software/spaceranger-4.0.1' >> ~/.bashrc
source ~/.bashrc

3. 下载对应物种的转录组参考数据集并解压

# Download mouse reference
curl -O https://cf.10xgenomics.com/supp/spatial-exp/refdata-gex-mm10-2020-A.tar.gz# Expected output
% Total    % Received % Xferd  Average Speed   Time    Time     Time  CurrentDload  Upload   Total   Spent    Left  Speed
1 9835M    1  158M    0     0  34.1M      0  0:04:48  0:00:04  0:04:44 34.1Mtar -xzvf refdata-gex-mm10-2020-A.tar.gz && rm refdata-gex-mm10-2020-A.tar.gz

下载对应物种的探针集：https://www.10xgenomics.com/support/software/space-ranger/downloads#reference-downloads

4. 准备自己的数据集，解压 FASTQ 文件，解压后会得到一堆 .fastq.gz 文件，例如：

datasets/V1_Adult_Mouse_Brain_fastqs/
├── V1_Adult_Mouse_Brain_S5_L001_R1_001.fastq.gz
├── V1_Adult_Mouse_Brain_S5_L001_R2_001.fastq.gz
├── ...

5. 运行

查看一下spaceranger count参数：

biostack@biostack:~$ spaceranger count -help
Count gene expression and protein expression reads from a single capture areaUsage: spaceranger count [OPTIONS] 
--id <ID> 
--create-bam <true|false> 
<--image <IMG>|--darkimage <IMG>|--colorizedimage <IMG>|--cytaimage <IMG>>……
--custom-bin-size <NUM>Bin Visium HD data to the specified bin size in addition to the standard binning size (2 um, 8 um, 16 um).Also produces a Loupe file and secondary analysis outputs at the specified bin size in addition to thestandard analysis bin size (8 um) if it is larger than 8 um
……

--id <ID>和--create-bam <true|false>是必须的，--image、--darkimage、--colorizedimage、--cytaimage 四选一，一般选择--cytaimage，默认输出2 um, 8 um, 16 um的结果。

运行：

spaceranger count --id=hd_count \--transcriptome=/path/to/refdata-gex-GRCh38-2020-A \--fastqs=/path/to/fastq \--probe-set=/path/to/Visium_Human_Transcriptome_Probe_Set_v2.0_GRCh38-2020-A.csv \--slide=H1-YD7CDZK \--area=A1 \--cytaimage=/path/to/CAVG10539_2023-11-16_14-56-24_APPS115_H1-YD7CDZK_A1_S11088.tif \--image=/path/to/APPS115_11088_rescan_01.btf \--create-bam=true/false

输出文件：