Old Links Used in Class - Spring 2026

Lecture 11 (3/11) RNA-Seq Analysis

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. Obtaining counts for genes

  • Perform transcript counting with stringtie count job file
  • Observe the files in the "stringtie_counts" folder.

    • Submit the job file


  • Old - Perform transcript counting with cufflinks job file

    • C. Identify differentially expressed genes (DEGs)

  • Download the counts_stringtie.txt file to the RNA-Seq folder
  • Download, modify, and submit DESeq.sbatch

  • old - Identify DEGs with the CuffDiff job file .
  • A pipeline that streamline the whole process. Example

    • Lecture 9/10 (3/9) High Throughput Sequencing (HTS) data analysis

    A.) Mapping to reference genome

  • Observe - Star Aligner explained.
  • Check to see if the file size are right, run FASTQ QC if necessary.
  • Observe the flybase FTP server
  • Download genome sequence and and the GTF files
  • Formate genome with the script file - try edit in your local text editor and upload with OOD
  • 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

    • Lecture 8/9 (3/4) Protein strcture and HTS data

    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.

  • Submit your protein for prediction AlphaFold3 server (requires registration - non-commercial only).

    • B. View and manipulating protein structture

  • Generate alpha helical wheel with Net Wheels .
  • PyMol for viewing and annotating 3D structure. Example 3NE4 & P01009.

    • C. Download HTS dataset from GEO to HiPerGator

  • Example - GSE62580 ; links for SRA dataset - SRP049144 - use the run selector to get accession list
  • Initiate UF OOD . Following log in, make a bookmark for quick access later.
  • 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 7 (3/2) Phylogenetic analysis and HTS data

    A.) Phylogenetic analysis

    Lecture 6 (2/27) Protein motifs, domains, & families

    A - Search for binary patterns

  • Make a /pattern project folder in your /GMS6014 folder
  • Download both the Bagua program and datafile dfile to the same folder dfile . ** only works in PC **
  • Search for Binary pattern such as "CXXC" or "[EDQN]X[^RKH]D[AST]"

    • B - Protein profile (protein family) databases:

  • Pfam at InterPro and the entry for IL6 ,
  • Prosite entry for IL6 ,

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


    C - Identifying motifs shared by a group of protein

  • Load the 8IL6.txt to MEME . 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

    • Blast output vs. motif search using Mast or Using BGHMM

    ** MEME, MAST, etc. can be run as a standalone program following installation . More options will be available to tailor the analysis. **

    Lecture 5 (2/25) 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)

    •     Blosum 62 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

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

    Local Alignment Web Service EMBOSS_Water or (backup LALIGN ).

    1.) alpha (gap opening penalty)=15, beta (extension penalty)=3; and
    2.) alpha=5, beta=1.

    Observe the results.


    links for previous lectures

    Lecture 4 (2/23) Pulblic Resources - Tools

    A.) Web-based tools

  • picking QPCR primer for your gene at Primer 3. You may use a sequence of interest to you or the human IL6

    • B.) Download and isntall the standalone NCBI-Blast: Manual at NCBI

  • Before installatin, read instruction for Windows, Mac.
  • Download the .win64.exe file for Windows or the .dmg for Mac from the NCBI ftp server . Pay attention to where the program is to be istalled. You may follow the default installation path but make a note of it as you may need it later.
  • Open a Powershell (Windows) or Terminal (Mac), type "makeblastdb" to verify the installation.

    • C.) Runnign blast

    1. With File Exporer (window) or Finder (Mac), navigate to your GMS6014 folder and make a new /blast folder
    2. Open a Powershell (Windows) or Terminal (Mac) in the blast folder, list the directory, then make new subfolders "dbs", "query", "out".
    3. 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.
    4. Search and download 3 IL6 proteins sequences in FASTA format from UniProt and save as "3IL6.txt" in the blast/query/ folder . example
    5. Formate the dataset for search by running "makeblastdb -in dbs/Dm -dbtype nucl". Check that by observing the new files generated in dbs/
    6. Run tblastn to search for orthologs of IL6 in Dm6.44.Allgenes

    Lecture 3 Resources for Bioinformatics (I) (2/20)

    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.

    • Information on human IL-6 at Reactome ; the example of an interaction involving IL6.

    Lecture 2 (2/18) - HiPerGator

    Tutorials:

  • HiperGator tutorial and introduction ;
  • Linux Command line tutorial.

    • Lecture 1 (2/16)

    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*. 

      3. *: 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.

      4. * 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: