教程：http://www.reibang.com/p/f6da86489784
http://www.reibang.com/p/07593e4d99a9
新版10X Visium
分析所要用到的
兩個軟件：Space Ranger - 1.0.0 (November 25, 2019) 和 Loupe Browser 4.0.0 (December 2, 2019)
Space Ranger
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spacerangeranger mkfastq包裝了Illumina的bcl2fastq制恍，解復(fù)用屡穗，并轉(zhuǎn)換barcode和read data為FASTQ* spaceranger count從
spaceranger mkfastq中獲取明場切片圖像和FASTQ文件严望，并執(zhí)行對齊，組織檢測杖剪，基準(zhǔn)檢測和條形碼/ UMI計數(shù)。該管道使用Visium空間條形碼生成特征點矩陣 feature-spot matrices，確定聚類并執(zhí)行基因表達(dá)分析。
這些管道將Visium專用算法與廣泛使用的RNA序列比對軟件STAR相結(jié)合凫乖。輸出以標(biāo)準(zhǔn)BAM，MEX弓颈，CSV拣凹，HDF5，TIFF恨豁，PNG，JPEG和HTML格式提供爬迟，并增加了空間信息橘蜜。
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捕獲點 -這些是載玻片上的不可見點，其中包含用于捕獲poly-adenylated mRNA的特殊寡核苷酸。* 基準(zhǔn)點fiducial spots：圍繞每個捕獲區(qū)域的帶有特殊圖案的點的框架计福。這些斑點可幫助樣本顯微學(xué)家查看放置組織的位置跌捆，Space Ranger還可使用這些斑點來確定圖像中捕獲區(qū)域的位置。* 字形glyphs-捕獲區(qū)域每個角上的基準(zhǔn)點的子集象颖，這些基準(zhǔn)點具有易于識別的形狀：沙漏佩厚，三角形，空心六邊形说订，實心六邊形抄瓦。* H＆E染色：-將蘇木精和曙紅施用于組織以突出組織結(jié)構(gòu)的過程。蘇木精使細(xì)胞核呈藍(lán)色陶冷，曙紅使細(xì)胞質(zhì)和細(xì)胞外基質(zhì)呈粉紅色钙姊。* 樣本 -應(yīng)用于Visium玻片上單個區(qū)域或由此得出的數(shù)據(jù)的單個組織切片。* 玻片序列號slide serial number -每個Visium玻片標(biāo)簽上印刷的唯一標(biāo)識符埂伦。序列號以“ V1”開頭煞额，并以短劃線和三位數(shù)字結(jié)尾，例如123沾谜。* 雙重索引dual indexing -一種通過使用兩個寡核苷酸序列對同一流動池flowcell上的多個樣品進行測序的策略膊毁，一個寡核苷酸序列連接到要測序的每個片段的任一末端，以便唯一地識別樣品基跑。Visium庫構(gòu)造僅使用此雙索引策略支持多路復(fù)用樣本婚温。請參閱下面的樣本索引。* 庫（或測序庫）-從單個載玻片區(qū)域制備的Visium空間條形碼測序庫涩僻。* 樣本索引 -用于文庫構(gòu)建的寡核苷酸序列缭召，用于區(qū)分在同一流通池上測序的多個樣本。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庫的構(gòu)造僅支持“雙重索引”（請參見上文）逆日。* sequencing run (or flowcell):一次測序儀器運行的輸出數(shù)據(jù)嵌巷，包括Illumina BCL文件∈页椋可以按泳道或樣本索引對數(shù)據(jù)進行多路分解搪哪。有關(guān)解復(fù)用的更多信息，請參見 mkfastq坪圾。
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此處的spaceranger鵬哥已經(jīng)在27上下載好了晓折，直接添加環(huán)境變量，然后source就行
index與cellranger用的是一樣的
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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
其中添加鵬哥的spaceranger的時候那個環(huán)境變量出 了問題
把最后的那個刪掉在source就好了
/data/yangpp/Space_Ranger/spaceranger-1.2.0/spaceranger
解決方法/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)
安裝成功
3.1. 下載簡單的CSV布局文件：spaceranger-tiny-bcl-simple-1.0.0.csv兽泄。
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. 下載Illumina實驗管理器樣本表：spaceranger-tiny-bcl-samplesheet-1.0.0.csv漓概。
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]
對于大多數(shù)測序?qū)嶒灒ㄗh使用簡單的csv樣本表病梢。簡單的csv格式只有三列（通道胃珍，樣本梁肿，索引），因此不太容易出現(xiàn)格式錯誤觅彰。您可以在中看到一個示例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
使用簡單布局mkfastq在tiny-bcl測序運行中運行的方法：
如果未按樣本索引測序吩蔑，則需要使用此格式。spaceranger-tiny-bcl-samplesheet-1.0.0.csv在運行管道之前簡要查看一下填抬。
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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后續(xù)：http://www.reibang.com/p/68a7655b4ba6
Seurat 新版教程：分析空間轉(zhuǎn)錄組數(shù)據(jù)
如何使用Seurat v3.2分析空間解析的RNA-seq數(shù)據(jù)
·歸一化
· 降維與聚類
· 檢測spatially-variable特性
· 交互式可視化
·與單細(xì)胞RNA-seq數(shù)據(jù)集成
· 處理多個片（multiple slices）
使用來自10x Genomics 的Visium技術(shù)（Visium technology）生成的數(shù)據(jù)集
