EasyAmplicon.pptx Script

[Audio] EasyAmplicon Pipeline Protocol for Streamline 16S RNA-Amplicon Sequencing Analysis Requirements To Run the Pipeline.

[Audio] What is the microbiome? Microbiome refers to the ecological group of microorganisms that study the symbiosis or pathology of animals and plants. The microbiome includes bacteria, archaea, fungi, protozoa and viruses. Studies have shown that it is very important in the host's immunity, metabolism sm and hormones. The synonym Microbiome includes microorganisms and their genomes..

[Audio] Animals and plants live in environments full of microorganisms Biological research is incomplete without microbes.

[Audio] 16S rDNA / rRNA gene (gene). 16S rDNA / rRNA gene (gene).

[Audio] Comparability / Repeatability 16S: Different variable regions produce different results The amplification efficiency and variation degree of different segments are different; how to compare the sequences of different segments? Different sampling methods, storage methods, DNA extraction, and sequencing platforms lead to different results Sample handling significantly affect community results DNA extraction has the greatest impact, and different sequencing platforms have amplification, sequencing, and error preferences ； Complex workflow and plethora of tools Difficulty evaluating microbiome tools (lack of microbiome standards) Difficult to create artificial samples of well-defined composition (massively unculturable) Use simulated populations and simulated data (too simplistic, artificially mixed).

[Audio] Commonly used sequencing platforms in this field generation sequence Second generation sequencing Sanger Illumina *Seq DNBSEQ-T7 Third generation sequencing Pacific Biosciences Nanopore.

[Audio] Microbiome Research Methods. Microbiome Research Methods.

[Audio] - Answerable Scientific Questions Culture omics - Bacteria can be cultured Amplicons------what are prokaryotic (bacterial/archaeal 16S) or eukaryotic (fungi/host/protozoan 18S/ITS), and their relative abundance Metagenome - what are the functional genes Metatranscriptome - which genes are being expressed Macroviridae - what kinds of DNA/RNA viruses are there Metaproteome - which genes are translated into proteins Metabolome - what kind of metabolites are there Metagenomics - Those Weird Modifications on DNA/RNA Single bacterial genome - what genes does a strain have? Pan-genomes—similarities and differences between a bacterium and its relatives.

[Audio] General Overview of our pipeline workflow.

[Audio] Operating system：recommended Windows10+ 64bit version(mainstream compatibility is good)， 32-bit systems are not recommended 、Mac Minimum Configuration：CPU dualcore, memory 4G, hard disk 10GB Recommended configuration ：CPU quad-core+, memory 8G+, hard disk 30GB+ space Common Software List Data analysis environment Shell + R +Integrated Development Environment： GitForWindows, Rstudio EasyAmplicon, EasyMicrobiome STAMP for (Difference Analysis and Visualization) Cytoscape、Gephi (Network Analysis and Visualization) Adobe Illustrator (Picture layout) Win subsystem +QIIME 2.

[Audio] Install dependent software Windows users(accounting for 87.5% of PCs) as an example, fast download GitForWindows, R, RStudio, STAMP v2.1.3 Install EasyAmplicon EasyAmplicon process(positive control) https://github.com/YongxinLiu/EasyAmplicon EasyMicrobiome software and database https://github.com/YongxinLiu/EasyMicrobiome Method 1. Visit the above link, click Code -- Download Method 2. Under Baidu Netdisk Load: https://pan.baidu.com/s/1Ikd_47HHODOqC3Rcx6eJ6Q?pwd=0315.

[Audio] Analysis Environment: GitForWindows (Only for Win users to install) Provides an environment for Windows to run Shell commands, which can be used in RStudio's Terminal. Official Website：http://gitforwindows.org/, click Download the latest version. Right-click the installation package Git-2.x.x.x-64-bit.exe to run as an administrator, and install according to the default parameters. (If unable to invoke, add C:\Program Files\Git\usr\bin to the Path in this computer's properties: Advanced > Environment Variables > System Variables）For specific use, see: Windows easily implements the Linux shell environment: ：gitforwindows Data analysis environment: R language and Rtools, an R package editing tool R language is currently the most popular statistical analysis language in the fields of biology and economics. official website：https://www.r-project.org/ Download the latest version：Downad CRAN China Tsinghua - Download R for Windows(Mac) —— base —— Download R 4.x.x Rtools in the same list as base, download Rtools There is an R-4.x.x-win.exe installer in the Win folder Double click the installer There is a Mac system installation package R-4.x.x.pkg in the Mac folder.

[Audio] R/Shell programming environment—RStudio Download page：https://posit.co/download/rstudio-desktop/ Choose the version that suits your system (Windows 10+ or macOS 10.14+), Download the latest version of the installer, or use the pre-downloaded installer RStudio-RStudio2022.12.0-353.exe for Win or RStudio-2022.12.0-353.dmg for Mac in the folder Right click and install as administrator Open it after completion, if there are multiple versions of R, you will be prompted to choose Select the first item - Use the system default R64 version –click OK That is, using the latest 64-bit version of the R locale installed..

[Audio] Switch the encoding in numerical order, close the file, exit the program, then reopen RStudio and review the article to restore normalcy..

[Audio] Installing R packages Access https://mirrors.tuna.tsinghua.edu.cn/CRAN/ - Download R for Windows – Rtools - RTools 4.2 - Rtools42 installer 4.3.zip package contains hundreds of commonly used R packages. Copy the compressed file 4.3.zip to the win-library directory. Select 4.3.zip, right-click, and choose 'Extract to current folder.' If prompted to replace files, you can choose 'Yes' for all. Note: Make sure not to have another 4.3 folder within the 4.3 folder, as it can cause issues with the system not being able to locate the packages.

[Audio] Install R Packages For Mac users, extract the compressed folder from the "Mac" directory to your Downloads folder. After that, execute the following command cp -r ~/Downloads/library/* /Library/Frameworks/R.framework/Versions/4.3/Resources/library/ Installing R packages—— Main menu to install R package---- CRAN official package Configure the CRAN mirror closest to you Easy and fast download (optional).

[Audio] Install packages from CRAN Enter the name of the package to be installed Install packages from CRAN. Install the R package in the code---- CRAN / Bioconductor / Github Install CRAN packages (the italic part is the name of the package, which can be replaced. Note: the devtools package is used to install the R packages from GitHub sources). install.packages("devtools) # New Method to Install Bioconductor Packages (R Version >=3.5. Note: edgeR is the most popular R package for sequencing data differential comparison） if (!requireNamespace("BiocManager", quietly = TRUE)) install.packages("BiocManager") BiocManager::install("edgeR") # Installing R Packages from GitHub (Italicized portion is the package name, replace it. Note: GitHub package names consist of both the username and the package name) library(devtools) devtools::install_github("microbiota/amplicon").

[Audio] Using Shell commands in RStudio Select Git Bash in RStudio's Tools menu - Global Options - Terminal, click OK Use Rstudio to open the shell.sh file in the Linux directory, cursor to process the code line or select the code to be executed, click the Run button (Run)/pressCtrl+Enter to execute the following code # Enter the C drive cd /c # create a directory mkdir -p amplicon # view file list ls If you don't see the Terminal or accidentally closed it, you can open it as shown in the following picture. Tools —— Terminal —— New Terminal (The shortcut is Alt+Shift+R).

[Audio] Microbiome Difference Comparison STAMP STAMP Result display STAMP is a software for analyzing microbial classification and functional spectrum. The latest version is 2.1.3. In the Downloads section, you can download the software that suits your Windows/Linux/MacOS version. STAMP can be compatible with different platforms, realizing Beta diversity scatter plots, species abundance histograms, box plots, and Post-hoc plots to display differential bacteria. You can also draw histograms with error bars, combined error bars and column separation charts, correlation scatter plots, density histograms, p-value histograms, etc. http://kiwi.cs.dal.ca/Software/STAMP Download the corresponding version of the system. There is a Windows version installation package STAMP_2_1_3.exe in the public\win directory.

[Audio] USEARCH / VSEARCH download 2.1 Already Integrated into EasyMicrobiome Download USEARCH Access http://www.drive5.com/usearch/download.html , Select USEARCH 10 version to download ： Windows：usearch10.0.240_win32.exe.gz Mac OS: usearch10.0.240_i86osx32.gz Download the file, extract it, and rename it as "usearch.exe". If you're a Windows user, save the file in the directory "C:/EasyMicrobiome/win/ Download VSEARCH https://github.com/torognes/vsearch On the homepage, go to the "Releases" section on the right side to find the latest download link, such as "vsearch-2.15.2-win-x86_64.zip After downloading, extract the file "vsearch.exe" and place it in the directory "C:/EasyMicrobiome/win/ ) Amplicon analysis workflow QIIME 2 installation Windows 10 system needs to install the Ubuntu 20.04 program in the application store. Please refer to Windows 10 to install the Linux subsystem Ubuntu 20.04LTS, easy to use bio-information software, and kill virtual machines efficiently Essentially, QIIME doesn't support Windows natively. Howeve, You can use it directly in the Ubuntu program interface or select "Bash (Windows Subsystem for Linux)" in the Terminal of RStudio to use QIIME 2. Next, install Conda in the Ubuntu/Mac environment, download the latest version of QIIME 2 software list, and install QIIME 2 with conda..

[Audio] QIIME 2 installation Amplicon analysis workflow QIIME 2 installation Linux subsystem of Windows 10 needs to be configured above Download, install and start conda wget -c https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh bash Miniconda3-latest-Linux-x86_64.sh -b -f ~/miniconda3/condabin/conda init Store the installation package public on the C drive (/mnt/c/EasyMicrobiome/linux/qiime22022.11.tar.gz) mkdir -p ~/miniconda3/envs/qiime2-2022.11 tar -xzf /mnt/c/db/conda/qiime2-2022.11.tar.gz -C ~/miniconda3/envs/qiime2-2022.11 conda activate qiime2-2022.11 conda unpack.

[Audio] Xshell Installation and use Xshell is a powerful terminal under Windows. Linux and Mac can directly use the ssh command to access the server The latest version download address： https://www.netsarang.com/zh/free-for-home-school/ Installation: Note that the type of Home/School can be used for free Run: For the first time, you need to configure the server information, file - new - name YSX host IP - OK - enter the user name and check the remember - enter the password and check the record – OK Terminal in RStudio can also access the server ssh username@serverIP Such as ssh [email protected].

[Audio] Xshell use. Xshell use.

[Audio] Installation and use FileZilla Official website：https://filezilla-project.org/ Download FileZilla Client download the latest version of the installation package suitable for your system. Configure Open the software - icon 'Open Site Manager' in the upper left corner - new site - name it YSX - host fill in 192.168.1.130 (IP address, fill in your own server or temporary IP for class), port 22; protocol SFTP, login type is common, user name The name is spelled in full, and the default password is ysx_amplicon. For the first connection choose Always Trust, OK..

[Audio] EditPlus Plain Text Editor: https://www.editplus.com/ edit plus plain text editor which is small size, fast startup, complete functions, can be opened and saved remotely, support for regular expression matching and replacement, support for displaying spaces and tabs, download the latest version 5.0 from the official website, or install public/win/epp510_1828_64bit.exe Registration code: Vovan 3AG46-JJ48E-CEACC-8E6EW-ECUAW Server Communication——Xshell & FileZilla  Xshell is a terminal used to log in to the server. Fill in your name and email to get the free version download link https://www.netsarang.com/zh/free-for-home-school/， Xshell doesn't run user alternative PuTTY properly http://www.putty.be/ putty.exe  FileZilla can continue uploading, downloading, and uploading data to NCBI. download link https://filezilla-project.org/download.php?type=client Unable to run this software. Alternatives: WinSCP://winscp.net/eng/download.php or Xftp  Simply complete the installation with default settings. Xshell requires selecting a license type as shown in the illustration to use it for free (the interface may be in English).

[Audio] Network Analysis and Visualization Cytoscape  Cytoscape is a graphical display network software, commonly used in biology to analyze the interaction between transcription factors and genes or proteins and proteins, GO and KEGG enrichment analysis  Software download address：http://www.cytoscape.org  Users who have not installed the Java runtime environment, first install jdk11.0.7_windows-x64_bin.exe  Download the installer Cytoscape_3_8_2_windows_64bit.exe under the folder or click the link  According to the default parameters to complete the installation  Cytoscape use video tutorial ：https://ke.qq.com/course/261290.

[Audio] Adobe Illustrator Picture beautification and typesetting  "AI" for short, is a very good vector graphics processing tool and image layout tool  It is a paid software developed by Adobe, here we provide you with a cracked free installation package for learning use only  Download the installation package under the folder or click the link Adobe_Illustrator_CC_2018_v22.1.0.314_x64_zh_CN_Portable.7z, After decompression, run the Illustratоr.exe program to use it (if you can't open it, try to run it as an administrator or App\Illustrator\Support Files\Contents\Windows\Illustrator.exe)  Use the video tutorial ：https://ke.qq.com/course/261607  You can experience the web version before installation ： https://mp.weixin.qq.com/s/0IfurkWazDj_gXVItSly6w.