+Annotation, which is the first stage, is an important task for
+extracting gene features. Indeed, to extract good gene feature, a good
+annotation tool is obviously required. The extraction of gene feature, the next stage, can be anything like gene names, gene sequences, protein sequences, and so on. Our method considers gene names, gene counts, and gene sequence for extracting core genes and producing chloroplast evolutionary tree. The final stage allows to visualize genomes and/or gene evolution in chloroplast. Therefore we use representations like tables, phylogenetic trees, graphs, etc. to organize and show genomes relationships, and thus achieve the goal of representing gene
+evolution. In addition, comparing these representations with ones issued from another annotation tool dedicated to large population of chloroplast genomes give us biological perspectives to the nature of chloroplasts evolution. Notice that a local database linked with each pipe stage is used to store all the informations produced during the process.
+
+\input{population_Table}
+
+\subsection{Genome annotation techniques}
+
+To obtain relevant annotated genomes, two annotation techniques from NCBI and Dogma are used. For the first stage, genome annotation, many techniques have been developed to annotate chloroplast genomes. These techniques differ
+from each others in the number and type of predicted genes (for example: \textit{Transfer RNA (tRNA)} and \textit{Ribosomal RNA (rRNA)} genes). Two annotation techniques from NCBI and Dogma are considered to analyze chloroplast genomes.
+
+\subsubsection{Genome annotation from NCBI}
+
+The objective is to generate sets of genes from each genome so that
+genes are organized without any duplication. The input is a list of
+chloroplast genomes annotated from NCBI. More precisely, all genomes
+are stored as \textit{.fasta} files which consists in a collection of
+protein coding genes\cite{parra2007cegma,RDogma} (gene that produce
+proteins) organized in coding sequences. To be able build the set of
+core genes, we need to preprocess these genomes
+using \textit{BioPython} package \cite{chapman2000biopython}. This
+step starts by converting each genome from FASTA file format to
+GenVision \cite{geneVision} format from DNASTAR. Each genome is thus
+converted in a list of genes, with gene names and gene counts. Gene
+name duplications can be accumulated during the treatment of a genome.
+These duplications come from gene fragments (\emph{e.g.} gene
+fragments treated with NCBI) and from chloroplast DNA sequences. To
+ensure that all the duplications are removed, each list of gene is
+translated into a set of genes. Note that NCBI genome annotation
+produces genes except \textit{Ribosomal (rRNA)} genes.
+
+\subsubsection{Genome annotation from Dogma}
+
+Dogma stands for \textit{Dual Organellar GenoMe Annotator}. It is an
+annotation tool developed at University of Texas in 2004 for plant
+chloroplast and animal mitochondrial genomes. This tool has its own
+database for translating a genome in all six reading frames and
+queries the amino acid sequence database using
+BLAST \cite{altschul1990basic} (\emph{i.e.} Blastx) with various
+parameters. Protein coding genes are identified in an input genome
+using sequence similarity of genes in Dogma database. In addition in
+comparison with NCBI annotation tool, Dogma can produce
+both \textit{Transfer RNAs (tRNA)} and \textit{Ribosomal RNAs (rRNA)},
+verify their start and end positions. further more, there is no gene duplication with gene annotations from Dogma after applying gene de-fragmentation process. In fact, genome annotation with Dogma can be the key difference when extracting core genes.
+
+The Dogma annotation process is divided into two tasks. First, we
+manually annotate chloroplast genomes using Dogma web tool. The output
+of this step is supposed to be a collection of coding genes files for
+each genome, organized in GeneVision file. The second task is to solve
+the gene duplication problem and therefore we have used two
+methods. The first method, based on gene name, translates each genome
+into a set of genes without duplicates. The second method avoid gene
+duplication through a defragment process. In each iteration, this
+process starts by taking a gene from gene list, searches for gene
+duplication, if a duplication is found, it looks on the orientation of
+the fragment sequence. If it is positive it appends directly the
+sequence to gene files. Otherwise reverse complement operations are
+applied on the sequence, which is then also append to gene files.
+Finally, a check for missing start and stop codons is performed. At
+the end of the annotation process, all the genomes are fully
+annotated, their genes are defragmented, and gene counts are
+available.
+
+\subsection{Core genes extraction}
+
+The goal of this stage is to extract maximum core genes from sets of
+genes. To find core genes, the following methodology is applied.
+
+\subsubsection{Preprocessing}
+
+In order to extract core genomes in a suitable manner, the genomic
+data are preprocessed with two methods: on the one hand a method based
+on gene name and count, and on the other hand a method based on a
+sequence quality control test.
+
+In the first method, we extract a list of genes from each chloroplast
+genome. Then we store this list of genes in the database under genome
+nam and genes counts can be extracted by a specific length command.
+The \textit{Intersection Core Matrix}, described in next subsection,
+is then computed to extract the core genes. The problem with this
+method can be stated as follows: how can we ensure that the gene which
+is predicted in core genes is the same gene in leaf genomes? The
+answer to this problem is that if the sequences of any gene in a
+genome annotated from Dogma and NCBI are similar with respect to a
+given threshold, the method is operational when the sequences are not similar. The problem of attribution of a sequence to a gene in the core genome come to light.
+
+The second method is based on the underlying idea that it is possible to predict the the best annotated genome by merging the annotated genomes from NCBI
+and Dogma according to a quality test on genes names and sequences. To
+obtain all quality genes of each genome, we consider the following
+hypothesis: any gene will appear in the predicted genome if and only
+if the annotated genes in NCBI and Dogma pass a specific threshold
+of \textit{quality control test}. In fact, the Needle-man Wunch
+algorithm is applied to compare both sequences with respect to a
+threshold. If the alignment score is above the threshold, then the
+gene will be retained in the predicted genome, otherwise the gene is
+ignored. Once the prediction of all genomes is done,
+the \textit{Intersection Core Matrix} is computed on these new genomes
+to extract core genes, as explained in Algorithm \ref{Alg3:thirdM}.
+
+\begin{algorithm}[H]
+\caption{Extract new genome based on gene quality test}
+\label{Alg3:thirdM}
+\begin{algorithmic}
+\REQUIRE $Gname \leftarrow \text{Genome Name}, Threshold \leftarrow 65$
+\ENSURE $geneList \leftarrow \text{Quality genes}$
+\STATE $dir(NCBI\_Genes) \leftarrow \text{NCBI genes of Gname}$
+\STATE $dir(Dogma\_Genes) \leftarrow \text{Dogma genes of Gname}$
+\STATE $geneList=\text{empty list}$
+\STATE $common=set(dir(NCBI\_Genes)) \cap set(dir(Dogma\_Genes))$
+\FOR{$\text{gene in common}$}
+ \STATE $g1 \leftarrow open(NCBI\_Genes(gene)).read()$
+ \STATE $g2 \leftarrow open(Dogma\_Genes(gene)).read()$
+ \STATE $score \leftarrow geneChk(g1,g2)$
+ \IF {$score > Threshold$}
+ \STATE $geneList \leftarrow gene$
+ \ENDIF
+\ENDFOR
+\RETURN $geneList$
+\end{algorithmic}
+\end{algorithm}
+
+\textbf{geneChk} is a subroutine used to find the best similarity score between
+two gene sequences after applying operations like \textit{reverse}, {\it complement},
+and {\it reverse complement}. Algorithm~\ref{Alg3:genechk} gives the outline of
+geneChk subroutine.
+
+\begin{algorithm}[H]
+\caption{Find the Maximum Similarity Score between two sequences}
+\label{Alg3:genechk}
+\begin{algorithmic}
+\REQUIRE $g1,g2 \leftarrow \text{NCBI gene sequence, Dogma gene sequence}$
+\ENSURE $\text{Maximum similarity score}$
+\STATE $score1 \leftarrow needle(g1,g2)$
+\STATE $score2 \leftarrow needle(g1,Reverse(g2))$
+\STATE $score3 \leftarrow needle(g1,Complement(g2))$
+\STATE $score4 \leftarrow needle(g1,Reverse(Complement(g2)))$
+\RETURN $max(score1,score2,score3,score4)$
+\end{algorithmic}
+\end{algorithm}
+
+\subsubsection{Intersection Core Matrix (\textit{ICM})}
+
+To extract core genes, we iteratively collect the maximum number of
+common genes between genomes and therefore during this stage
+an \textit{Intersection Core Matrix} (ICM) is built. ICM is a two
+dimensional symmetric matrix where each row and each column correspond
+to one genome. Hence, an element of the matrix stores
+the \textit{Intersection Score} (IS): the cardinality of the core
+genes set obtained by intersecting one genome with another
+one. Maximum cardinality results in selecting the two genomes having
+the maximum score. Mathematically speaking, if we have $n$ genomes in
+local database, the ICM is an $n \times n$ matrix whose elements
+satisfy:
+\begin{equation}
+score_{ij}=\vert g_i \cap g_j\vert
+\label{Eq1}
+\end{equation}
+\noindent where $1 \leq i \leq n$, $1 \leq j \leq n$, and $g_i, g_j$ are
+genomes. The generation of a new core gene depends obviously on the
+value of the intersection scores $score_{ij}$. More precisely, the
+idea is to consider a pair of genomes such that their score is the
+largest element in ICM. These two genomes are then removed from matrix
+and the resulting new core genome is added for the next iteration.
+The ICM is then updated to take into account the new core gene: new IS
+values are computed for it. This process is repeated until no new core
+gene can be obtained.
+
+We can observe that the ICM is very large due to the amount of
+data. As a consequence, the computation of the intersection scores is
+both time and memory consuming. However, since ICM is a symetric
+matrix we can reduce the computation overhead by considering only its
+triangular upper part. The time complexity for this process after
+enhancement is thus $O(\frac{n.(n-1)}{2})$. Algorithm ~\ref{Alg1:ICM}
+illustrates the construction of the ICM matrix and the extraction of
+the core genes, where \textit{GenomeList} represents the database
+storing all genomes data. At each iteration, it computes the maximum
+core genes with its two genomes parents.
+
+% ALGORITHM HAS BEEN REWRITTEN
