空间转录组

分析空间转录组数据学习笔记

2020-11-26  本文已影响0人  jiarf

教程：https://www.jianshu.com/p/f6da86489784
https://www.jianshu.com/p/07593e4d99a9
新版10X Visium

分析所要用到的

两个软件：Space Ranger - 1.0.0 (November 25, 2019) 和 Loupe Browser 4.0.0 (December 2, 2019)

Space Ranger

这些管道将Visium专用算法与广泛使用的RNA序列比对软件STAR相结合。输出以标准BAM，MEX，CSV，HDF5，TIFF，PNG，JPEG和HTML格式提供，并增加了空间信息。


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wget http://cf.10xgenomics.com/supp/spatial-exp/spaceranger-tiny-bcl-1.0.0.tar.gz
10:39:56 jiarongf@172.16.10.223:/data1/jiarongf/Visium/learn
$
tar -xvzf spaceranger-tiny-bcl-1.0.0.tar.gz
10:44:50 jiarongf@172.16.10.223:/data1/jiarongf/Visium/learn
$
ls
spaceranger-tiny-bcl-1.0.0  spaceranger-tiny-bcl-1.0.0.tar.gz
10:44:59 jiarongf@172.16.10.223:/data1/jiarongf/Visium/learn
$
ls spaceranger-tiny-bcl-1.0.0
Data  InterOp  _src

其中添加鹏哥的spaceranger的时候那个环境变量出 了问题
把最后的那个删掉在source就好了
/data/yangpp/Space_Ranger/spaceranger-1.2.0/spaceranger

解决方法/data/yangpp/Space_Ranger/spaceranger-1.2.0

11:07:33 jiarongf@172.16.10.223:/data1/jiarongf/Visium/learn
$
spaceranger
spaceranger spaceranger-1.2.0
Process 10x Genomics Spatial Gene Expression data

USAGE:
    spaceranger <SUBCOMMAND>

FLAGS:
    -h, --help       Prints help information
    -V, --version    Prints version information

SUBCOMMANDS:
    count               Count gene expression and feature barcoding reads from a single capture area
    aggr                Aggregate data from multiple 'spaceranger count' runs
    targeted-compare    Analyze targeted enrichment performance by comparing a targeted sample to its cognate parent WTA sample (used as input for targeted gene expression)
    targeted-depth      Estimate targeted read depth values (mean reads per spot) for a specified input parent WTA sample and a target panel CSV file
    mkfastq             Run Illumina demultiplexer on sample sheets that contain 10x-specific sample index sets
    testrun             Execute the 'count' pipeline on a small test dataset
    mat2csv             Convert a gene count matrix to CSV format
    mkref               Prepare a reference for use with 10x analysis software. Requires a GTF and FASTA
    mkgtf               Filter a GTF file by attribute prior to creating a 10x reference
    upload              Upload analysis logs to 10x Genomics support
    sitecheck           Collect linux system configuration information
    help                Prints this message or the help of the given subcommand(s)

安装成功

3.1. 下载简单的CSV布局文件：spaceranger-tiny-bcl-simple-1.0.0.csv。

11:04:02 jiarongf@172.16.10.223://data1/jiarongf/Visium/learn
$
wget http://cf.10xgenomics.com/supp/spatial-exp/spaceranger-tiny-bcl-simple-1.0.0.csv
--2020-11-23 11:13:57--  http://cf.10xgenomics.com/supp/spatial-exp/spaceranger-tiny-bcl-simple-1.0.0.csv
Resolving cf.10xgenomics.com (cf.10xgenomics.com)... 104.18.0.173, 104.18.1.173, 2606:4700::6812:1ad, ...
Connecting to cf.10xgenomics.com (cf.10xgenomics.com)|104.18.0.173|:80... connected.
HTTP request sent, awaiting response... 301 Moved Permanently
Location: https://cf.10xgenomics.com/supp/spatial-exp/spaceranger-tiny-bcl-simple-1.0.0.csv [following]
--2020-11-23 11:13:57--  https://cf.10xgenomics.com/supp/spatial-exp/spaceranger-tiny-bcl-simple-1.0.0.csv
Connecting to cf.10xgenomics.com (cf.10xgenomics.com)|104.18.0.173|:443... connected.
HTTP request sent, awaiting response... 200 OK
Length: 41 [text/csv]
Saving to: ‘spaceranger-tiny-bcl-simple-1.0.0.csv’

spaceranger-tiny-bcl-simple-1.0.0.csv                       100%[========================================================================================================================================>]      41  --.-KB/s    in 0s

2020-11-23 11:13:58 (5.58 MB/s) - ‘spaceranger-tiny-bcl-simple-1.0.0.csv’ saved [41/41]

4.1. 下载Illumina实验管理器样本表：spaceranger-tiny-bcl-samplesheet-1.0.0.csv。

wget http://cf.10xgenomics.com/supp/spatial-exp/spaceranger-tiny-bcl-samplesheet-1.0.0.csv
--2020-11-23 11:15:13--  http://cf.10xgenomics.com/supp/spatial-exp/spaceranger-tiny-bcl-samplesheet-1.0.0.csv
Resolving cf.10xgenomics.com (cf.10xgenomics.com)... 104.18.1.173, 104.18.0.173, 2606:4700::6812:ad, ...
Connecting to cf.10xgenomics.com (cf.10xgenomics.com)|104.18.1.173|:80... connected.
HTTP request sent, awaiting response... 301 Moved Permanently
Location: https://cf.10xgenomics.com/supp/spatial-exp/spaceranger-tiny-bcl-samplesheet-1.0.0.csv [following]
--2020-11-23 11:15:13--  https://cf.10xgenomics.com/supp/spatial-exp/spaceranger-tiny-bcl-samplesheet-1.0.0.csv
Connecting to cf.10xgenomics.com (cf.10xgenomics.com)|104.18.1.173|:443... connected.
HTTP request sent, awaiting response... 200 OK
Length: 552 [text/csv]
Saving to: ‘spaceranger-tiny-bcl-samplesheet-1.0.0.csv’

spaceranger-tiny-bcl-samplesheet-1.0.0.csv                  100%[========================================================================================================================================>]     552  --.-KB/s    in 0s

2020-11-23 11:15:15 (10.0 MB/s) - ‘spaceranger-tiny-bcl-samplesheet-1.0.0.csv’ saved [552/552]

对于大多数测序实验，建议使用简单的csv样本表。简单的csv格式只有三列（通道，样本，索引），因此不太容易出现格式错误。您可以在中看到一个示例spaceranger-tiny-bcl-simple-1.0.0.csv：

11:19:34 jiarongf@172.16.10.223://data1/jiarongf/Visium/learn
$
head spaceranger-tiny-bcl-simple-1.0.0.csv
Lane,Sample,Index
1,test_sample,SI-TT-D9

使用简单布局mkfastq在tiny-bcl测序运行中运行的方法：

如果未按样本索引测序，则需要使用此格式。spaceranger-tiny-bcl-samplesheet-1.0.0.csv在运行管道之前简要查看一下。

11:20:36 jiarongf@172.16.10.223://data1/jiarongf/Visium/learn
$
head spaceranger-tiny-bcl-samplesheet-1.0.0.csv
[Header],,,,,,,,
IEMFileVersion,4,,,,,,,
Investigator Name,user,,,,,,,
Experiment Name,hiseq_test,,,,,,,
Date,12/2/19,,,,,,,
Workflow,GenerateFASTQ,,,,,,,
Application,HiSeq FASTQ Only,,,,,,,
Assay,TruSeq HT,,,,,,,
Description,hiseq sample sheet,,,,,,,
Chemistry,Default,,,,,,,
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后续：https://www.jianshu.com/p/68a7655b4ba6

Seurat 新版教程：分析空间转录组数据

如何使用Seurat v3.2分析空间解析的RNA-seq数据
·归一化
· 降维与聚类
· 检测spatially-variable特性
· 交互式可视化
·与单细胞RNA-seq数据集成
· 处理多个片（multiple slices）
使用来自10x Genomics 的Visium技术（Visium technology）生成的数据集

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