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\title{Bioinformatics - Sequence Alignments and DB Searching}
\author{Dr. Gautam B. Singh }
\date{}

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\begin{document}

\maketitle


\begin{enumerate}
    \item \textbf{Basics}: Download the myoglobin protein sequences from the pig, chicken, and king cobra (snake). The accession numbers for these sequences are: \verb+NP_999401+, \verb+NP_001161224+, and \texttt{ETE72530}, respectively. Since these are full protein sequences, we will use the global sequence alignment method (the Needleman-Wunch). The Matlab function to perform this alignment is \textit{nwalign}. Here is an example of the usage of this function: the sequence \textit{seq1} and \textit{seq2} are being aligned using the PAM250 scoring matrix. The function returns the score and the alignment.
    \begin{verbatim}
        [score, align] = nwalign(seq1, seq2, 'scoringmatrix', @pam250)
    \end{verbatim}
    Perform the global alignments using PAM-10 and PAM-250 and complete the similarity matrix shown in Fig.~\ref{fig:dist_matrix}. Note that the similarity matrix is symmetric, so only the upper half needs to be computed. 
    
    \begin{enumerate}
        \item Complete the following matrix:
            \begin{figure}[ht]
            \centering
            \includegraphics{DistanceMatrix-Orthologs}
            \caption{Similarity Scores Matrix}
            \label{fig:dist_matrix}
            \end{figure}
        \item Is a snake more like a chicken or a pig? 
        \item Is a chicken more like a pig or more like a snake?
        \item Are the results consistent across the PAM-10 and PAM-250 matrices?
        \item Why are the results with PAM-10 higher than PAM-250?
    \end{enumerate}
    
    
    \item \textbf{The H and N Proteins}: Next, we will look at the efficacy of flu vaccines. Influenza, commonly known as the flu, is caused by a virus that attacks the upper respiratory tract (i.e., the nose, the throat, and the lungs). Cold and dry weather allows the virus to survive longer outside the body than in warm weather. 
    
    There are three types of influenza viruses: A, B, and C. Type A can infect humans, other mammals, and birds and can spread fast. Types B and C only affect humans, and type C causes only mild infections. Type A viruses are sub-typed based on proteins on the virus's surface, specifically proteins hemagglutinin (H or HA) and neuraminidase (N or NA).  Types A and B viruses continually evolve genetically with changes to the amino acid sequence of the H and N proteins, thus eluding the host's immune system. 
    
    Each year, the influenza vaccine is designed using the prevalent strains from previous years. Generally, the vaccine contains three virus strains: two type A strains and one type B strain. A Type C strain is not used since it only causes a mild infection. Each February, the World Health Organization (WHO) picks the three influenza virus strains to be included in the vaccine for that year.
    
    To find information on the strains in that season's influenza vaccine, visit the  Center for Disease Control \verb+ https://www.cdc.gov/fluview/+ and look at ``Past Weekly Surveillance Reports'' to select the previous influenza season. 
    
    %We will be investigating the 2009-10 influenza season. So, you will need to look at the 2008-09 influenza season summary and read the section titled ``Composition of the 2009-2010 Influenza Vaccine."
    
    WHO recommended that the 2009--10 Northern Hemisphere influenza vaccine contain 
    \begin{enumerate}
    \item \texttt{A/Brisbane/59/2007}-like (H1N1), 
    \item \texttt{A/Brisbane/10/2007}-like (H3N2), and 
    \item \texttt{B/Brisbane/60/2008}-like (B/Victoria lineage) viruses. 
    \end{enumerate}
    
    \textit{Antigenic Characterization for 2009 - 2010}: A flu pandemic in the Northen Hemisphere during 2009--10. 
    
    CDC antigenically characterized 2 seasonal influenza A (H1N1), 14 influenza A (H3N2), 43 influenza B, and 1,904 \textit{pandemic} H1N1 viruses. However, 99.5\% of \emph{pandemic} H1N1 viruses were the \texttt{A/California/07/2009} (H1N1) reference virus.\footnote{The 2 seasonal influenza A (H1N1) viruses tested were related to \texttt{A/Brisbane/59/2007}. The 14 influenza A (H3N2) viruses tested were related to \texttt{A/Brisbane/10/2007}. Of the 43 influenza B viruses tested, 38 (88.4\%) were related to B/Brisbane/60/2008, with the remaining five (11.6\%) viruses tested belonging to the B/Yamagata lineage.}
    
    \begin{enumerate}
    	\item Search the GenBank and find the Accession Numbers for the three virus strains used to develop the 2009--10 vaccine. 
	
		\item Find the Accession Number for \texttt{A/California/07/2009} that caused the pandemic.
	
        \item Compare the sequence of the three 2009-10 vaccine viruses to \texttt{A/California/07/2009} virus.  Specifically, a pairwise comparison of the hemagglutinin protein and the neuraminidase protein should be performed. Overall, how similar are the hemagglutinin and neuraminidase protein sequences of the \texttt{A/California/07/2009} to the three virus strains used in creating the vaccines for that season?\footnote{ A 2009 pandemic H1N1 vaccine based on \texttt{A/California/07/2009} had to be created that season.}
        
        \item Based on sequence similarity, how well do you think the vaccine protected a vaccinated person in the Northern Hemisphere was protected in the 2009--2010 season?
    \end{enumerate}
    
    \item \textbf{BLAST} We've cloned a new mouse cDNA with an open reading frame. We want to use BLAST  \verb+https://blast.ncbi.nlm.nih.gov/+ to characterize this mRNA.
    \begin{verbatim}
    MKRKFVGAAIGGALAVAGAPVALSAVGFTGAGIAAGSIAAKMMSAAAIANGGGIAAGGLVATLQSVGVLGL
    STITNIILVAVGTATGARAEGSMGASREQESGPQDPPQELQEPQEPPSCKKQDLNLGKFVGAAIGGALAVA
    GAPIALSAVGFTGAGIAAGSIAAKMMSAAAIANGGGIAAGGLVATLQSVGILGLSTSTNIILGAVGAATGA
    TAAGAMGACREQEPGLQDLQQEPKEPQEPQELQKQQEPQEPQELQKQQETQETQETQELQKTQEPPSYEK
    \end{verbatim}
    \begin{enumerate}
        \item[$\ddag$] Configure BLAST to run a search using all of the following parameters: \textbf{NR} database,  using the \textbf{blastp} algorithm, with the filter on, and using =the matrix \textbf{blosum62}. 
        \item What is the purpose of running the search with a filter?
        \item From the graphic output, what conserved domain is in our protein in the region that it spans?
        \item How many hits are there?  How many hits (from the list) have an E-score of &lt; 0.05? How many hits (from the list) have 0.05 &lt; E-score &lt; 1.00?
        \item Provide the accession numbers, descriptions, and E-scores for the five best target sequences. 
        \item Review the taxonomy report. To which organism do the hits belong?
        \item Now run the search \textit{without} the filter. How do the answers to the above questions change? 
        \item Look at the top hit and enter the RefSeq entry. What is its accession number? What species is it from? How long is it? What is its name? According to the Genbank record, from where to where is the domain that blast found?
        \item Download this GenBank RefSeq to a local file and compute its alignment with our sequence using the MATLAB \textit{nwalign} program. Include the alignment with your answer. Is this a true hit? Why or why not? 
    \end{enumerate}
    
    
\end{enumerate} 


\end{document}
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