Old Links Used in Class - Spring 2023
Lecture 13 Genome browser and genomic sequence analysis (3/10)
A.) Displaying genomic dataset with UCSC browser
UCSC Genome Browser is a popular server for viewing genomic data -- pay attention to select the right genome release. Sign up with the service will allow you to save the analysis sessions and share the results with others. Example
Download and install the IGB genome browser, which you can run on your own computer
An example of a saved session , which will persist and you can send the link to collaborators
B.) How to obtain genomic sequence surrounding your gene?
* From genome browser - example
* For gneomes not available in public genome browser - use Fastacmd or your own Pyhon script
C.) Identifying TF and epigenetic regulator binding sites in the genome
Searching for TF binding
sites in the DNA
sequence using TFsiteScan or
Lecture 12 Intro to R and machine learning (3/8)
The R project and R studio download site.
Demo R code: General ;
for tidyverse ; and machine learning.
Lecture 11 RNA-Seq (3/6)
A.) Identify differentially expressed genes (DEGs)
Observe the output of cufflinks.
Compile the assembly files for Whole Gut
Identify DEGs with a job file samples
A pipeline that streamline the whole process. Example
B.) Functional interpretation of HTS (RNA-Seq) data
Our analysis of change of gene expression in old vs young tissues produced CuffDiff Output (save the file and view with excel, use sort to extract list of DEGs based on p/q value or the significance call)
Enrichment analysis .
Try the g:profiler analysis with the list of significantly (p less than 0.0001) increased genes we generated with Cuff_Diff.
Lecture 10 RNA-Seq (3/3)
A. RNA_seq data analysis overview.
Review paper 1 and 2 .
Examples of protocols: 1.) Tophat-->Cofflink--Cuffdiff ;
2.) hisat2-->StringTie. You could also make your own based on the needs of your project with available components
B.) Mapping to reference genome
- Check to see if the file size are right, run FASTQ QC if necessary.
- Map to the reference genome using the script/job file StarMap . Edit the script/job file with your text editor and load to your RNA-Seq directory.
- submit the job file
Perform transcript counting with cufflinks job file
Intro for Star Aligner
- If you just got your HiPerGator account with the class, simply do "sbatch SCRIPT_FILE"
- If you had your primary account with your group, you have the option of using your group account, which is the default, or the class account by specifying "--account=gms6014 --qos=gms6014".
Lecture 9 High throughput sequencing (HTS) data analysis (3/1)
A.) Obtaining HTS dataset from GEO
Example - GSE62580 ; links for SRA dataset - SRP049144 - use the run selector to get accession list
using fastq-dump, you may choose to either download 4 samples of whole gut samples into a /wg folder within your working directory
for batch download, using a .sbatch job file - download the example ; change it to your email address. upload it to your folder and submit the job with "sbatch --account=gms6014 --qos=gms6014 [Filename]
Lecture 8 Protein struture, AlphaFold, and HiPerGator (2/27)
A.) Predicting protein structure
Alphafold2 paper by Deepmind .
Introduction to the Alphafold project on the Deepming website.
PDB - Protein Data Bank ( wwPDB ), played a fundamental role in improving protein struture analysis.
B.) Running AF2 on Hipergator
- Obtain a fasta format file of the protein that you are interested to predict structure. Save and load it to your folder in /gms6014/share/YourName/AF2. (Prtoein file we use before.)
- Edit the Script file on your local computer:
upload it to the same AF2 folder as above.
- change email address on the #SBATCH --mail-user line.
- change the output folder name.
- change the name of the fasta formate file.
- Open terminal connect to HiPerGaot. Navigate to your own AF2 folder, run the command "sbatch --account=gms6014 --qos=gms6014 AlphaFold2.sh".
- Obtain sequences in fasta format. example
- Load sequence into ClustalOmega and perform global alignment; then Draw N-J tree.
- Example of the output ; Click the "phylogenetic Tree" tab to view the tree and downlaod the tree file to your GMS6014/phylogenetics/ folder
- Observe and manipulate the tree with Phylodendron
- example of trees 1 and 2 ;
- Standalone ClutalOmega for downlaod
- Comprehensive protein analysis tool JalView
Lecture 6 - Protein Domains and Motifs (2/22)
A. More on scoring matrices
The orginal Henikoff and Henikoff paper that served as the fundation of using BLOSUM62 as the default for BLAST search
Comparison of PAM vs BLOSUM matrices
B.) Limitations of generic scoring matrices
Search for binary patterns
Extra: Search for Binary pattern with Bagua in dfile.
Identifying motifs shared by a group of protein - load the 8IL6.txt to
Leave your email address for accessing the results.
--> Shared motifs identified by MEME.
an example of scoring matrix generated by MEME
The Motifs identified by MEME could then be used for searching database of sequences
vs. motif search using Mast or Using BG_0.5
C.) Protein Profile (protein family) Databases:
Pfam at InterPro and the entry for IL6 ,
Prosie entry for IL6 ,
Search the Blast hit and the motif search hit against
Pfam or Prosite
Lecture 5 (2/20) Alignment and sequence similarity
1.) How did BLAST identify the hit(s) for us?
Blast results from standalone blast - IL6_Dm6.44Genes and IL6_Dm6.32.cDNAs .
What is Blast ? - Basic Local Alignment Search Tool ( NCBI site ; Nature Education page ;)
The basis of quantifying sequence similarity
- Block Substituion Matrices (BloSuM)
matrix, Blosum matrices.
What is block?
"Many known proteins can be grouped into families according to functional and sequence similarities. The similarity of the proteins across the sequences in each family is far from uniform. While some regions are clearly conserved, others display little sequence similarity. Often the conserved regions are crucial to the protein's function, for example enzymatic catalytic sites. Such conserved regions can be used to probe an uncharacterized sequence to indicate its function. " -- Pietrokovski, Henikoff, Henikoff 1996 Link
2.) How scoring matrix and penalty affect the outcome of local alignment
two test sequences , perform local alignment with the following parameters.
Local Alignment Web Service EMBOSS_Water or
- Different matrices- try local aligment with either Blosum62 or
Blosum 35 - observe the difference.
- Different gap penalties- with matrix set to blosum62, try:
(gap opening penalty)=15, beta (extension penalty)=3; and
Observe the results.
Lecture 4 (2/17) Standalone Applications
1.) Download and isntall the standalone NCBI-Blast: manual at NCBI
Before installatin, read instruction for Windows,
Download the .win64.exe file for Windows or the .dmg for Mac from the NCBI ftp server . Change the default installation path to YourHomeFolder/GMS6014/
Open a Command (Windows) or Terminal (Mac), navigate to the blast folder, list subfolders, then make new subfolder "dbs", "query", "out".
Download Data set for BLAST search. Genomic dataset can be downloaded at Ensemble. A previously downloaded dataset - All Genes in D. mel genome in FASTA format
- save the data set in blast/dbs.
Download 3 IL6 proteins sequences from UniProt and save as "3IL6.fasta" in the blast/query/ folder .
2.) Runnign blast
- Open a Command (Windows) or Terminal (Mac), navigate to the blast folder, list the directory, then make new subfolders "dbs", "query", "out".
- Download Data set for BLAST search. Genomic dataset can be downloaded at Ensemble. A previously downloaded dataset - All Genes in D. mel genome in FASTA format. Name this file as Dm6.44.AllGenes and save it in blast/dbs.
- Search and download 3 IL6 proteins sequences in FASTA format from UniProt and save as "3IL6.txt" in the blast/query/ folder . example
- Formate the dataset for search by running "makeblastdb -in dbs/Dm -dbtype nucl". Check that by observing the new files generated in dbs/
- Run tblastn to search for orthologs of IL6 in Dm6.44.Allgenes
Lecture 3 (2/15) HiPerGator
1.) Presentation slides for class.
HiperGator tutorial and introduction ;
Linux Command line tutorial.
Lecture 2 (2/13)
A.) List of public resources. ·
B.) Navigate the web of information on your favorite gene (or IL6)
Search for the gene in Gene vs. Protein database
Observe the difference between all text search and advacned search
Pay attention to the multitude of links associated the Gene entry.
Compare the human "IL6" entry in the NCBI Gene database v.s. the EBI UniProt database.
Pathway and interaction information on human IL-6 at
C.) Web-based tools
picking QPCR primer for your gene at Primer 3
D.) Linux Environment
Log into HiperGator using your gatorLink credential and DHO - navigate to course folder
Make a directory with your first name in the /blue/gms6014/share/ folder. This folder will allow me to view your progress and help touble-shooting.
make a soft link from you home directory "~" to your course folder "ln -s /blue/gms6014/share/yourfirstname/ gms6014 "
make a shell file and run it.
Lecture 1 (2/10)
A.) Retrieve and save sequence file:
- Try different view (format) of the same entry by selecting
different "display settings".
- Download the FASTA files of nucleotide and protein sequence into your local
*: make a
folder such as "GMS6014" in your home fodler. Save all
course-related files in this folder. Avoid space in folder and file names
- Open the saved file using a text editor such as Notepad in Wondows and TextEdit in MacOS.
* consider using a dedicated text editor for bioinformatics projects. Such as NotePad++ (for Windos only) or
Emacs (all major OS)
B.) Local storage of sequence files:
- Use .doc or .rtf files for formatting and annotation of
sequences. example (Right click the
link and save in the "C:\temp\GMS6014" folder)
- Use .seq or .fasta to store raw sequences in fasta format for downstream
analysis. example (Right click the
link and save in the "C:\temp\GMS6014" folder)
- Change folder option to view file extension. Change association to always open .seq or .txt files in your designated text editor
- Try to load the MySequence.txt to Webcutter or NEBCutter for identifying
restriction sites; then try to load the MySequence.doc file.
C. Familiar yourself with the HiPerGator System if you have not use it