实验记录1：安装Cellranger与Seurat，下载数据

2018-9-21

梗概

1. 安装单细胞测序数据处理软件Cellranger
2. 安装R包Seurat
3. 下载cellranger参考数据Human reference (GRCh38) dataset与Human Cell Alters（HCA）的Ischaemic Sensitivity of Human Tissue数据

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准备工作

登录实验室服务器

1. 下载Cellranger及其参考数据

Cell Ranger是一组分析工具，用于处理Chromium单细胞RNA-seq输出来整理读数，生成基因-细胞矩阵以进行聚类和基因表达分析。 Cell Ranger包括与单细胞基因表达实验相关的四个主要工具：

1. cellranger mkfastq
   它将Illumina序列发生器生成的raw base call（BCL）文件解构为FASTQ文件。它是Illumina的bcl2fastq的包装器，具有10x库特有的其他有用功能和简化的样本表格式。

2. cellranger count
   takes FASTQ files from cellranger mkfastq and performs alignment, filtering, barcode counting, and UMI counting. It uses the Chromium cellular barcodes to generate gene-barcode matrices, determine clusters, and perform gene expression analysis. The count pipeline can take input from multiple sequencing runs on the same library.

3. cellranger aggr
   aggregates outputs from multiple runs of cellranger count, normalizing those runs to the same sequencing depth and then recomputing the gene-barcode matrices and analysis on the combined data. The aggr pipeline can be used to combine data from multiple samples into an experiment-wide gene-barcode matrix and analysis.

4. cellranger reanalyze
   takes gene-barcode matrices produced by cellranger count or cellranger aggr and reruns the dimensionality reduction, clustering, and gene expression algorithms using tunable parameter settings.

下载Cellranger 2.2.0：
参考网站：
https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest

下载软件

curl -o cellranger-2.2.0.tar.gz "http://cf.10xgenomics.com/releases/cell-exp/cellranger-2.2.0.tar.gz?Expires=1537482514&Policy=eyJTdGF0ZW1lbnQiOlt7IlJlc291cmNlIjoiaHR0cDovL2NmLjEweGdlbm9taWNzLmNvbS9yZWxlYXNlcy9jZWxsLWV4cC9jZWxscmFuZ2VyLTIuMi4wLnRhci5neiIsIkNvbmRpdGlvbiI6eyJEYXRlTGVzc1RoYW4iOnsiQVdTOkVwb2NoVGltZSI6MTUzNzQ4MjUxNH19fV19&Signature=Dz1xfrWJWVjCtaXrqoMQ7MsUwgzvNAPC~W7sJS6yqhxUPh462hJWdVKm187Z7bqHHVBEhC0vWEZSJ~~vbABZ72X-mHunO0RRX0qNYIUNHI2ag0hH-BGsmC86flI0geB7swc5OYrHe92jTjDMYGj7gnafpT2GtJC-BlpDMy9gsCDYB-eDaU6--kK~Dvd4JHbCN9EaljLem1628~XN-LbQMDjF9AIqVVSfWDUKHAsE6DZxasbMrQOAj7VVKEXqsymQj8nc-x6nAZ~nevvUAs-a5ddvJ5oPPoDUTNA-Dkq1qQ5NnbOPq9wGKjWAUu7skQIm96JN867Ol8BuctzmQelZdg__&Key-Pair-Id=APKAI7S6A5RYOXBWRP

下载参考数据

curl -O http://cf.10xgenomics.com/supp/cell-exp/refdata-cellranger-GRCh38-1.2.0.tar.gz

解压cellranger和ref-data的压缩包

$ tar -xzvf cellranger-2.2.0.tar.gz
$ tar -xzvf refdata-cellranger-GRCh38-1.2.0.tar.gz

配置PATH

$ export PATH=/opt/cellranger-2.2.0:$PATH

2. 下载Seurat

运行R

install.packages('Seurat')

报错：

更换服务器

3. 下载Human Cell Atlas数据

Human Cell Atlas Preview Datasets数据为Human Cell Atlas（HCA）的第一批公开的单细胞测序数据库，HCA计划目的在于收集人类各个器官组织的单细胞测序数据，汇合成一个完整的人类单细胞图谱，便于后续人们对单个的组织的单细胞测序数据研究时的横向对比和参照。
目前一共有三个数据，分别为人类免疫细胞的普查数据、缺血敏感性人类脾脏组织和黑色素瘤浸润机制基质和免疫细胞的单细胞RNA-seq数据。
其中前两个数据集是通过10x genomics公司的测序方法测得，为fastq格式，正好可用于cell ranger进行整理和分析。
下载地址：
https://preview.data.humancellatlas.org

HCA网站所给出的数据信息

安装pip（老师完成）

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下载方式：

1. Download the shell script for the dataset you are interested in

Click on the download datasete button to save a shell script that will download data.

2. Run the script in the terminal

After saving the script, run it in yuor terminal as follows:

chmod +x /download/hca-download-ischaemic-sensitivity 

Then, run:

/download/hca-download-ischaemic-sensitivity 

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先下载了Ischaemic Sensitivity of Human Tissue
（因为这个数据集小，另外一个熟知了流程后再下载）
Assessment of ischaemic sensitivity of human spleen tissue by single cell RNA-seq.
点击“Primary Data”