Old Links Used in Class - Spring 2024

Lecture 12 (3/8)

A.) Merge gtf files from individual samples to prepare for comparison.

B.) transfer files to and from HiPerGator

• Observe FileZila.
• Globus web file manager is an easy alternative:
1. set up account with Globus
2. Follow the instruction at here to access files in HiPerGator.
3. Download a genes.fpkm_tracking file for observing on your laptop.

C.) Displaying genomic dataset with UCSC browser

D.) Identify differentially expressed genes (DEGs)

Lecture 11 (3/6)

A.) Mapping to reference genome

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

* Identifyng genome-wide trancription factor binding sits with ChIP-Seq data analysis. An example of ChIP-Seq data and TF binding sites prediction

Lecture 10 RNA-Seq (3/4)

A.) Obtaining HTS dataset from GEO

B. RNA_seq data analysis overview.

C.) Mapping to reference genome

1. Check to see if the file size are right, run FASTQ QC if necessary.
2. Make genome index file accordiing to genome sequence and gtf files and script file
3. 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.
4. submit the job file
• 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 "sbatch --account=gms6014 --qos=gms6014".

Lecture 9 Phylogenetic analysis and HTS data (3/1)

A.) Phylogenetic analysis

• Jalview for multiple alignment and phylogenetic analysis
• Practice:
1. Obtain sequences in fasta format. example
2. Load sequence into ClustalOmega and perform global alignment; then Draw N-J tree.
3. For more options, install the standalone Clustalo
4. Example of the output ; Click the "phylogenetic Tree" tab to view the tree and downlaod the tree file to your GMS6014/phylogenetics/ folder
5. Observe and manipulate the tree with Phylodendron

• example of trees 1 and 2 ;
• Standalone ClutalOmega for downlaod
• Comprehensive protein analysis tool JalView

B.) Obtaining HTS dataset from GEO

Lecture 8 Protein struture (2/28)

A.) Predicting protein structure

B.) Running AF2 on Hipergator

1. 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.)
2. Edit the Script file on your local computer:
   1. change email address on the #SBATCH --mail-user line.
   2. change the output folder name.
   3. change the name of the fasta formate file.
   upload it to the same AF2 folder as above.
3. Open terminal connect to HiPerGaot. Navigate to your own AF2 folder, run the command "sbatch --account=gms6014 --qos=gms6014 AlphaFold2.sh".

Lecture 7 - Protein Domains and Motifs (2/26)

A - Search for binary patterns

B - Identifying motifs shared by a group of protein

Blast output vs. motif search using Mast or Using BG_0.5

C - Protein profile (protein family) databases:

Search the Blast hit and the motif search hit against Pfam or Prosite

Lecture 6 - Protein Domains and Motifs (2/23)

A litter more on scoring matrices

B - Global vs. Local alignment

Using the two test sequences , perform

(A) Local Alignment using EMBOSS_Water ; or

(B) Global alignment with Stretcher

Lecture 5 (2/21) Alignment and sequence similarity

1.) How did BLAST identify the hit(s) for us?

The basis of quantifying sequence similarity

    Blosum 62 matrix, Blosum matrices.

2.) How scoring matrix and penalty affect the outcome of local alignment

Using the two test sequences , perform local alignment with the following parameters.

Local Alignment Web Service EMBOSS_Water or (backup LALIGN ).

• Different matrices-  try local aligment with either Blosum62 or Blosum 35 - observe the difference.
• Different gap penalties- with matrix set to blosum62, try:  

1.) alpha (gap opening penalty)=15, beta (extension penalty)=3; and

2.) alpha=5, beta=1.

Observe the results.

Lecture 4 (2/19) Standalone Applications

1.) Download and isntall the standalone NCBI-Blast: manual at NCBI

2.) Runnign blast

1. Open a Command (Windows) or Terminal (Mac), navigate to the blast folder, list the directory, then make new subfolders "dbs", "query", "out".
2. 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.
3. Search and download 3 IL6 proteins sequences in FASTA format from UniProt and save as "3IL6.txt" in the blast/query/ folder . example
4. Formate the dataset for search by running "makeblastdb -in dbs/Dm -dbtype nucl". Check that by observing the new files generated in dbs/
5. Run tblastn to search for orthologs of IL6 in Dm6.44.Allgenes

Lecture 3 (2/15) HiPerGator

1.) Presentation slides for class.

2.) Tutorials:

Lecture 2 (2/14)

A.) Retrieve and save sequence file:

1. Try different view (format) of the same entry by selecting different "display settings".
2. Download the FASTA files of nucleotide and protein sequence into your local computer*. 

*: 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

3. 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.) List of public resources. ·

• EBI list of services,
• NCBI Site Map. ·
• Kyoto Encyclopedia of Genes and Genomes - KEGG.
• Deposite of genomes - ENSEMBL .
• Examples of species-specific database: Flybase, wormbase.

D.) Navigate the web of information on your favorite gene (or IL6)

Pathway and interaction information on human IL-6 at Reactome ;

Lecture 1 (2/12)

Familiar yourself with the HiPerGator System if you have not use it

• HiPerGator Tutorials

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