Cell Ranger是一个10X genomics公司的单细胞分析软件,将原始的fastq文件生成后续分析的feature-barcode表达矩阵。 其中包括很多模块,本次主要介绍cellranger mkfastq、cellranger count,cellranger aggr 和 cellranger reanalyze四个功能模块。 1.1 下载
进入cellranger官网(https://support./)后,发现支持的分析模块有很多,先介绍单细胞转录组。选择单细胞转录组模块,点击进入 软件-下载-选择你想要的cellranger版本, https://support./single-cell-gene-expression/software/downloads/latest 1)curl ,wget 和 直接网页下载,三种方式均可; 2)记得下载注释文件 3)注意查看md5值(很重要) 1.2 安装Step1:解压下载的软件安装包 #进入文件存放的位置,示例为opt $ cd /opt #解压 $ tar -xzvf cellranger-6.0.1.tar.gz解压缩到一个名为cellranger-6.0.1的新目录,包含Cell Ranger及其依赖项和Cell Ranger脚本。 Step2:同样的方式解压参考文件 $ tar -xzvf refdata-gex-GRCh38-2020-A.tar.gzStep3:配置环境 将Cell Ranger目录添加到$PATH中,注意路径要准确,示例为/opt , $ export PATH=/opt/cellranger-6.0.1:$PATH为使用方便可以添加到.bashrc文件中。 1.3 测试安装可以查看一下版本和帮助,或者参考官网的Site Check Script 的方式。 cellranger -V cellranger -h下载:https://support./single-cell-gene-expression/software/downloads/latest 安装:https://support./single-cell-gene-expression/software/pipelines/latest/installation cellranger使用mkfastq功能来拆分Illumina 原始数据(raw base call (BCL)),输出 FASTQ 文件。
2.1 下载示例数据点击下载即可 2.2 Running mkfastq with a Simple CSV Samplesheet1)首先示例矩阵数据解压缩,当前目录下生成cellranger-tiny-bcl-1.2.0文件夹 tar -xvzf cellranger-tiny-bcl-1.2.0.tar.gz2)Simple CSV Samplesheet文件 格式:三列(Lane、Sample、Index),逗号分隔,不太容易出现格式错误。示例数据cellrangerver -tiny-bcl-simple-1.2.0.csv如下: Lane,Sample,Index 1,test_sample,SI-TT-D9Lane | Which lane(s) of the flowcell to process. Can be either a single lane, a range (e.g., 2-4) or '*' for all lanes in the flowcell. | Sample | The name of the sample. This name is the prefix to all the generated FASTQs, and corresponds to the --sample argument in all downstream 10x pipelines. Sample names must conform to the Illumina bcl2fastq naming requirements. Only letters, numbers, underscores and hyphens area allowed; no other symbols, including dots (".") are allowed. | Index | The 10x sample index that was used in library construction, e.g., SI-TT-D9 or SI-GA-A1 |
3)run mkfastq 需要安装且配置bcl2fastq软件 $ cellranger mkfastq --id=cellranger-tiny-bcl-1.2.0 \ --run=/path/to/cellranger-tiny-bcl-1.2.0 \ --csv=cellranger-tiny-bcl-simple-1.2.0.csvid :即为解压后的文件夹名字 run:为解压后的文件夹的绝对路径 在id名的新文件夹中既有生成的fastq文件了,可以用于后续的count分析。 另一种请参考https://support./single-cell-gene-expression/software/pipelines/latest/using/mkfastq 此处使用转录组数据进行count分析,通过fastq文件得到细胞和基因的定量结果。 3.1 必要参数$ cellranger count --id=sample345 \ --transcriptome=/opt/refdata-gex-GRCh38-2020-A \ --fastqs=/home/jdoe/runs/HAWT7ADXX/outs/fastq_path \ --sample=mysample \ --expect-cells=1000 \--id= 名称 --fastqs= fastq.gz文件保存的绝对路径 --sample= fastq.gz文件名"-"之前的字段 --transcriptome= 参考基因组路径 --expect-cells= 期望细胞数(可选) 3.2 参数列表参数详细介绍详见: https://support./single-cell-gene-expression/software/pipelines/latest/using/count#args中的Command-Line Argument Reference 部分 可以注意下以下参数: --expect-cells | (optional) Expected number of recovered cells. Default: 3,000 cells. | 和实验匹配 | --nosecondary | (optional) Add this flag to skip secondary analysis of the feature-barcode matrix (dimensionality reduction, clustering and visualization). Set this if you plan to use cellranger reanalyze or your own custom analysis. | 仅获得表达矩阵,不进行后续的降维,聚类和可视化分析 | --chemistry | (optional) Assay configuration. NOTE: by default the assay configuration is detected automatically, which is the recommended mode. You should only specify chemistry if there is an error in automatic detection. Select one of: |
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3.3 结果文件结果文件列表以及简要描述说明 File Name | Description |
| web_summary.html | Run summary metrics and charts in HTML format | 网页简版报告以及可视化 | metrics_summary.csv | Run summary metrics in CSV format |
| possorted_genome_bam.bam | Reads aligned to the genome and transcriptome annotated with barcode information |
| possorted_genome_bam.bam.bai | Index for possorted_genome_bam.bam |
| filtered_feature_bc_matrix | Filtered feature-barcode matrices containing only cellular barcodes in MEX format. (In Targeted Gene Expression samples, the non-targeted genes are not present.) | 过滤掉的barcode信息 | filtered_feature_bc_matrix_h5.h5 | Filtered feature-barcode matrices containing only cellular barcodes in HDF5 format. (In Targeted Gene Expression samples, the non-targeted genes are not present.) | 过滤掉的barcode信息HDF5 format; | raw_feature_bc_matrices | Unfiltered feature-barcode matrices containing all barcodes in MEX format | 原始barcode信息 | raw_feature_bc_matrix_h5.h5 | Unfiltered feature-barcode matrices containing all barcodes in HDF5 format | 原始barcode信息HDF5 format | analysis | Secondary analysis data including dimensionality reduction, cell clustering, and differential expression |
| molecule_info.h5 | Molecule-level information used by cellranger aggr to aggregate samples into larger datasets |
| cloupe.cloupe | Loupe Browser visualization and analysis file | Loupe Cell Browser 输入文件 | feature_reference.csv | (Feature Barcode only) Feature Reference CSV file |
| target_panel.csv | (Targeted GEX only) Targed panel CSV file |
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参考资料:https://support./single-cell-gene-expression/software/pipelines/latest/using/mkfastq 精心整理(含图PLUS版)|R语言生信分析,可视化(R统计,ggplot2绘图,生信图形可视化汇总)
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