Counting chromosomes of Artemisia tridentata using root meristems of individual lines maintained in vitro at BSU. This approach will help ascertaining the ploidy level (2n) of our individual lines.
Day 1
Young whitish roots are sampled from individuals cultured in vitro either at Growth or Rooting phases.
Recipes to prepare solutions presented here were adapted from this document (in french).
For 1000 mL for acetocarmin solution:
This solution can be stored for a long time. Staining can be intensified by adding ferric chloride (FeCl2·6H2O); add 5 mL of a 10 % ferric chloride solution per 100 mL of % acetocarmine.
Kansas State University as also put together a slightly different protocol for the acetocarmin solution: https://www.k-state.edu/wgrc/electronic_lab/aceto_stain.html
Estimate 2C genome size of Artemisia tridentata based on leaf material using the one-step protocol with general purpose buffer (GPB) by Loureiro et al. (2007) and the protocol described in Pellicer and Leitch (2014). The publications are accessible here.
The flow cytometry will be conducted at BSU using a BD Accuri™ C6 Flow Cytometer.
One thing that we need to bear in mind is that the coefficient of variation of the fluorescence peaks should be below 5%, and that the measurements should be taken using a linear scale rather that a logarithmic scale.
Genome size is estimated using young, fresh leaves for both our target (sagebrush) and internal standards.
Concerning reference internal standards, we recommend that several species, covering a broad range of genome sizes, are kept growing in the laboratory to enable the most appropriate standard to be selected for each particular analysis.
Check what seeds we have in the lab.:
The recipe of the buffer and the steps are described in the paper, but briefly:
GBP: 0.5 mM spermine.4HCl, 30 mM sodium citrate, 20 mM MOPS (MOPS, 4-Morpholinepropane sulfonate), 80 mM KCl, 20 mM NaCl, 0.5 % (v/v) Triton X-100, pH 7.0.
The buffer is now ready to use. If you notice that the sample becomes dark when prepared, add 200 ul of B-mercaptoethanol to the buffer and mix (or equivalent if aliquots have been prepared).
To more efficiently benefit from our Linux computers, we will provide overview of protocols to:
In RStudio, you can send commands directly into the Terminal (instead of the Console) by using this combination of keys: Ctrl + Alt + Enter. The Terminal has to be selected before using this approach.
For this purpose, we will be using the adduser command:
#Create user, add password and create home directory (without admin rights)
sudo adduser username
#For example
sudo adduser anthonymeltonYou will be asked to answer multiple questions, only the password is mandatory.
Complete creation of sub-directories for new user by typing these commands:
#Switch to the new user account (see below for more details)
su username
#Create sub-directories (e.g. Documents/, Downloads/, etc.)
xdg-user-dirs-update --forceTo view the list of users on the Linux computer type this command:
cut -d : -f 1 /etc/passwdTo view the list of logged in users do:
wTo provide admin (sudo) rights to a user that you created do the following:
#Create a new used with admin/sudo rights
sudo usermod -aG sudo username
#For example
sudo usermod -aG sudo anthonymeltonOn the other hand, if you want to reverse a user from sudo to standard rights, do the following:
#General syntax
sudo deluser username sudoThis command will only remove user ‘username’ from the sudo group, but it will not delete the user permanently from the system. Now, they become regular user and cannot do any administrative tasks as sudo user.
After someone leaves the lab, you might have to delete their user account. You can do that by using the following command, but please make sure that you have archived all their data and code prior to proceeding.
#If you want to delete a user and its home directory and mail spool
sudo deluser --remove-home usernameTo create a group, here genomics type the following command:
#Create a group entitled `genomics`
sudo groupadd genomicsTo add users to the groups (here genomics) type the following command:
#Add users to the group, here `genomics`
sudo usermod -a -G genomics userID
# Examples
sudo usermod -a -G genomics svenbuerki
sudo usermod -a -G genomics bio_11
sudo usermod -a -G genomics anthonymeltonTo know which users are already members of a group type the following command:
grep genomics /etc/groupTo switch user on Ubuntu you need to type the following command:
#General syntax
su USER_name
#Example (you will have to provide password for bio_11)
su bio_11Here we use the ln command to make a symbolic link (or a kind of alias) to a program in the bin/. This means that the program will be accessible to anybody by typing a shortcut (or alias).
Since the symbolic link will reside in the bin/ folder, first you will be navigating to this location as follows:
#Navigate where the symbolic link should reside
cd /usr/local/bin/Then we will establish the symbolic link as follows:
#Use ln protocol to establish symbolic link
# pathtoprogram = path to executable program
# alias = the name you give to the symbolic link or alias
sudo ln -s pathtoprogram alias
#Example with R
sudo ln -s /home/bioinformatics/R-4.0.4/bin/R R-4In this example, everybody will be able to access R version 4.0.4 by typing R-4 in the Terminal.
Since NGS data are taking a lot of space on the hard drive, users might would like to quickly estimate the amount of space left on their hard drive (using df) or space taken by files (using du). To do that, please use the following commands:
The df protocol gives you insights into disk free space. To know about the free space on all disks do:
df -HTo get the same information, but for the disk specific to a folder (here /home), do:
df -H /homeThe du protocol gives you insights into disk usage. here we propose a command that gives you specifically the disk usage taken by files:
#Disk usage taken by files in a folder
du -h -s *Some jobs take a long time to run. If you want, you can add a command to send you an email upon completion of the job. Ue the following syntax:
command; echo "JOB COMPETED! Go online to check output." | mail -s "Job is completed" username@boisestate.eduYour email might be redirected into your SPAM box, so please check for it.
A Samba file server enables file sharing across different operating systems over a network. It lets you access your desktop files from a laptop and share files with Windows and macOS users. Here, we will be using this protocol to share our external hard drive remotely via smb protocol.
If it is not done yet, please install Samba on Ubuntu computer as follows:
sudo apt update
sudo apt install sambaWe can check if the installation was successful by running:
whereis sambamkdir) or identify a folder on your Ubuntu computer where the smb server will be connected to. In this example, we use our shared folder in /home/Genomics_shared and created a folder for smb protocols (smbshare).#Location where smb server will be mounted
/home/Genomics_shared/sambashare#1. Mount smb server
# adjust:
# - username
# - smb IP address
# - Location for mounting smb server on your computer
sudo mount -t cifs -o username=svenbuerki //cifs-prd-01/research/SvenBuerki /home/Genomics_shared/sambasharesudo protocol. Here is an example where we copy a folder from computer to mounted smb server:#2. Copy folder from Linux box to smb server
sudo cp -r IDT2-2_NovaSeq_Illumina_April2019/ /home/Genomics_shared/sambashare/If you are interested in setting-up a samba (smb) served from your Linux computer to be shared with Windows users follow this approach:
https://ubuntu.com/tutorials/install-and-configure-samba#1-overview
You might have to download NGS data from different providers (e.g. GeneWIZ) and they will set-up an sftp server for you. Here is a short tutorial on the procedure used to download data:
Open a Terminal and start a tmux session as follows:
tmuxThis will open a safe working environment that can be accessed very easily. Please read protocol on tmux sessions here.
To connect to an sftp server you need:
#General syntax
sftp username@sftp.IP
#Example with GeneWIZ
sftp anthonymelton_boisestate@sftp.genewiz.comThe get or mget protocols used to copy files over don’t allow to set the destinations path (where the files will be saved on your local computer), but you can easily set this by using the lcd command. Here is an example based on our project structure.
#Copy files from sftp server to /home/Genomics_shared/G1_b2_1/
lcd /home/Genomics_shared/G1_b2_1/The best practice is to copy the whole project folder over onto your local computer. This can be done by using get -r command. Here is an example with some GeneWIZ data.
#Copy whole project over
get -r 30-470086959Depending on the sive of the files (usually several Gb) it might take hours for the transfer to be completed. be patient!