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
-
spacerangeranger mkfastq包装了Illumina的bcl2fastq，解复用，并转换barcode和read data为FASTQ* spaceranger count从
spaceranger mkfastq中获取明场切片图像和FASTQ文件，并执行对齐，组织检测，基准检测和条形码/ UMI计数。该管道使用Visium空间条形码生成特征点矩阵 feature-spot matrices，确定聚类并执行基因表达分析。
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。
image.png
image.png
-
捕获点 -这些是载玻片上的不可见点，其中包含用于捕获poly-adenylated mRNA的特殊寡核苷酸。* 基准点fiducial spots：围绕每个捕获区域的带有特殊图案的点的框架。这些斑点可帮助样本显微学家查看放置组织的位置，Space Ranger还可使用这些斑点来确定图像中捕获区域的位置。* 字形glyphs-捕获区域每个角上的基准点的子集，这些基准点具有易于识别的形状：沙漏，三角形，空心六边形，实心六边形。* H＆E染色：-将苏木精和曙红施用于组织以突出组织结构的过程。苏木精使细胞核呈蓝色，曙红使细胞质和细胞外基质呈粉红色。* 样本 -应用于Visium玻片上单个区域或由此得出的数据的单个组织切片。* 玻片序列号slide serial number -每个Visium玻片标签上印刷的唯一标识符。序列号以“ V1”开头，并以短划线和三位数字结尾，例如123。* 双重索引dual indexing -一种通过使用两个寡核苷酸序列对同一流动池flowcell上的多个样品进行测序的策略，一个寡核苷酸序列连接到要测序的每个片段的任一末端，以便唯一地识别样品。Visium库构造仅使用此双索引策略支持多路复用样本。请参阅下面的样本索引。* 库（或测序库）-从单个载玻片区域制备的Visium空间条形码测序库。* 样本索引 -用于文库构建的寡核苷酸序列，用于区分在同一流通池上测序的多个样本。On the Illumina platform, these sequences are read out as separate "index reads" and reads are sorted into sample-specific files using mkfastq. The Visium library construction supports only "dual-indexing" (see above).Visium库的构造仅支持“双重索引”（请参见上文）。* sequencing run (or flowcell):一次测序仪器运行的输出数据，包括Illumina BCL文件。可以按泳道或样本索引对数据进行多路分解。有关解复用的更多信息，请参见 mkfastq。
image.png
此处的spaceranger鹏哥已经在27上下载好了，直接添加环境变量，然后source就行
index与cellranger用的是一样的
image.png
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,,,,,,,
image.png
image.png
image.png
image.png
image.png
image.png
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
