-The field of Genome annotation pay a lot of attentions where the ability to collect and analysis genomical data can provide strong indicator for the study of life\cite{Eisen2007}. A lot of genome annotation centres present various types of annotation tools (i.e cost-effective sequencing methods\cite{Bakke2009}) on different annotation levels. Methods of gene finding in annotated genome can be categorized as: Alignment-based, composition based or combination of both\cite{parra2007cegma}. The Alignment-based method is used when we try to predict a coding gene (i.e. Genes that produce proteins) by aligning DNA sequence of gene to the protein of cDNA sequence of homolog\cite{parra2007cegma}, this approache used in GeneWise\cite{birney2004genewise} with known splicing signals. Composition-based mothod (known as \textit{ab initio} is based on a probabilistic model of gene structure to find genes and/or new genes accoding to the probility gene value, this method like GeneID\cite{parra2000geneid}. In this section, we will consider a new method of finding core genes from large amount of chloroplast genomes, as a solution of the previous method where stated in section two. This method is based on extracting gene features. The question now is how can we have good annotation genome? To answer this question, we need to focusing on studying the annotation's accuracy (systematically\cite{Bakke2009}) of the genome. The general overview of the system is illustrated in Figure 1.\\
+The field of Genome annotation pays a lot of attentions where the ability to collect and analysis genomical data can provide strong indicator for the study of life\cite{Eisen2007}. Four of genome annotation centers, (such as, \textit{NCBI\cite{Sayers01012011}, Dogma \cite{RDogma}, cpBase \cite{de2002comparative}, CpGAVAS \cite{liu2012cpgavas}, and CEGMA\cite{parra2007cegma}}), present various types of annotations tools (i.e cost-effective sequencing methods\cite{Bakke2009}) on different annotation levels. Generally, one of three methods of gene finding in annotated genome can be categorized using these centers: \textit{alignment-based, composition based, or combination of both\cite{parra2007cegma}}. The alignment-based method is used when we try to predict a coding gene (i.e. genes that produce proteins) by aligning DNA sequence of gene to the protein of cDNA sequence of homology\cite{parra2007cegma}. This approache also is used in GeneWise\cite{birney2004genewise}. Composition-based mothod (known as \textit{ab initio}) is based on a probabilistic model of gene structure to find genes and/or new genes according to the probability gene value (GeneID\cite{parra2000geneid}). In this section, we will consider a new method of finding core genes from large amount of chloroplast genomes, as a solution of the problem resulting from the method stated in section two. This method is based on extracting gene features. A general overview of the system is illustrated in Figure \ref{Fig1}.\\
-\begin{figure}[H]
-\caption{A general overview of the system}
+\begin{figure}[H]
\centering
- \includegraphics[width=0.5\textwidth]{generalView}
+ \includegraphics[width=0.7\textwidth]{generalView}
+\caption{A general overview of the system}\label{Fig1}
\end{figure}
-In Figure 1, we illustrate the general overview of the system. In this system, there are three main stages: \textit{Database, Gene extraction ,} and \textit{relationships}. There are many international nucleotide sequence databases like (GenBank/NCBI in USA at (http://www.ncbi.nlm.nih.gov/genbank/),\\ EMBL-Bank/ENA/EBI in Europe at (http://www.ebi.ac.uk/ena/), and DDBJ in Japon at (http://www.ddbj.nig.ac.jp/)). In our work, the database must be any confident data source that store annotated or unannotated chloroplast genomes. We will consider GenBank/NCBI database as our nucleotide sequences database. Extract Gene Features, we refer to our main process of extracting needed information to find core genome from well large annotation genomes. Thanks to good annotation tool that lead us to extract good gene features. Here, Gene features can be anything like (genes names, gene sequences, protein sequence,...etc). To verify the results from our system, we need to organize and represent our results in the form of (tables, phylogenetic trees, graphs,...,etc), and compare these results with another annotation tool like Dogma\cite{RDogma}. All this work is to see the relationship among our large population of chloroplast genomes and find the core genome for root ancestral node. Furthermore, in this part we can visualize the evolution relationships of different chloroplast organisms.\\
-The output from each stage in our system will be considered to be an input to the second stage and so on. The rest of this section, in section 3.1, we will introduce some annotation problem with NCBI chloroplast genomes and we will discuss our method for how can we extract useful data. Section 3.2 we will present here our system for calculating evolutionary core genome based on another annotation tool than NCBI.
+In Figure 1, we illustrate the general overview of system pipeline: \textit{Database, Genomes annotation, Core extraction,} and \textit{relationships}. We will give a short discussion for each stage of the model in order to understand all core extraction process. This work starts with a gene Bank database; however, many international Banks for nucleotide sequence databases (such as, \textit{GenBank} \cite{Sayers01012011} in USA, \textit{EMBL-Bank} \cite{apweiler1985swiss} in Europe, and \textit{DDBJ} \cite{sugawara2008ddbj} in Japon) where exist to store various genomes and DNA species. Different Biological tools are provided to analyse and annotate genomes by interacting with these databases to align and extract sequences to predict genes. The database in this model must be taken from any confident data source that store annotated and/or unannotated chloroplast genomes. We will consider GenBank-NCBI \cite{Sayers01012011} database to be our nucleotide sequences database. Annotation (as the second stage) is considered to be the first important task for Extract Gene Features. Good annotation tool lead us to extracts good gene feature. In this paper, two annotation techniques from \textit{NCBI, and Dogma} are used to extract \textit{genes features}. Extracting Gene feature (as a third stage) can be anything like (genes names, gene sequences, protein sequence,...etc). Our methodologies in this paper consider gene names, genes counts, and gene sequences for extracting core genes and producing chloroplast evolutionary tree. \\
+In last stage, features visualization represents methods to visualize genomes and/or gene evolution in chloroplast. By using the forms of (tables, phylogenetic trees, graphs,...,etc) to organize and represent genomes relationships can achieve the goal of gene evolution with what the biological expert needs. In addition, compare these forms with another annotation tool forms for large population of chloroplast genomes give us biological perspective to the nature of chloroplasts evolution. \\
+A Local database attached with each pipe stage is used to store all the informations of extraction process. The output from each stage in our system will be an input to the second stage and so on.
\subsection{Genomes Samples}
-In this research, we retrieved 107 genomes of Chloroplasts from NCBI where 9 genomes considered as not good. These 99 genomes lies in the 11 types of chloroplast families, divided as 11 for Algues Brunes, 3 Algue Rouges, 17 Algues Vertes, 45 Angiospermes, 3 Brypoytes, 2 Dinoflagelles, 2 Euglenes, 5 Filicophytes, 7 Gymnosperms, 2 Lycophytes, and 1 Haptophytes, as show in Table 1.
-\begin{figure}[H]
-\caption{Sample Genomes with its Families}
- \centering
- \includegraphics[width=0.7\textwidth]{image1}
-\end{figure}
-\begin{figure}[H]
- \centering
- \includegraphics[width=0.7\textwidth]{image2}
-\end{figure}
+In this research, we retrieved genomes of Chloroplasts from NCBI. Ninety nine genome of them were considered to work with. These genomes lies in the eleven type of chloroplast families. The distribution of genomes is illustrated in detail in Table \ref{Tab2}.
-\subsection{Gene Extraction Techniques from annotated NCBI genomes}
-With NCBI, the idea is to use the existing annotations of NCBI with chloroplast genomes. To extract the core and pan genes: Core extraction techniques with NCBI are based on two techniques: Gene count and Gene contents based on some similarity issues.
+\input{population_Table}
-\subsubsection{Core genes based on NCBI Gene names and Counts}
-The trivial and simple idea to construct the core genome is based on the extraction of Genes names (as gene presence or absence). For instant, in this stage neither sequence comparison nor new annotation were made, we just want to extract all gene counts stored in each chloroplast genome then find the intersection core genes based on gene names.\\
+\subsection{Genome Annotation Techniques}
+Genome annotation is the second stage in the model pipeline. Many techniques were developed to annotate chloroplast genomes but the problem is that they vary in the number and type of predicting genes (i.e the ability to predict genes and \textit{for example: Transfer RNA (tRNA)} and \textit{Ribosomal RNA (rRNA)} genes). Two annotation techniques from NCBI and Dogma are considered to analyse chloroplast genomes to examine the accuracy of predicted coding genes.
-\textbf{Step I: pre-processing}\\
-The objective from this step is to organize, solve genes duplications, and generate sets of genes for each genome. The input to the system is a list genomes from NCBI stored as a \textit{.fasta} file that include a collection of coding genes\cite{parra2007cegma}(genes that produce protein) with its coding sequences.
-As a preparation step to achieve the set of core genes, we need to translate these genomes and extracting all information needed to find the core genes. This is not an easy job. The output from this operation is a lists of genes stored in a local database for genomes, their genes names and genes counts. In this stage, we will accumulate some Gene duplications with each genome treated. In other words, duplication in gene name can comes from genes fragments as long as chloroplast DNA sequences. Identical state, which it is the state that each gene present only one time in a genome (i.e Gene has no copy) without considering the position or gene orientation can be reached by filtering the database from redundant gene name. To do this, we have two solutions: first, we made an orthography checking. Orthography checking is used to merge fragments of a gene to be one gene so that we can solve a duplication.
-Second, we convert the list of genes names for each genome (i.e. after orthography check) in the database to be a set of genes names. Mathematically speaking, if $g=\left[g_1,g_2,g_3,g_1,g_3,g_4\right]$ is a list of genes names, by using the definition of a set in mathematics, we will have $set(g)=\{g_1,g_2,g_3,g_4\}$, where each gene represented only ones. With NCBI genomes, we do not have a problem of genes fragments because they already treated it, but there are a problem of genes orthography. This can generate the problem of gene lost in our method and effect in turn the core genes.
-The whole process of extracting core genome based on genes names and counts among genomes is illustrate in Figure 3.
+\subsubsection{genome annotation from NCBI}
+The objective from this step is to organize genes, solve gene duplications, and generate sets of genes from each genome. The input to the system is our list of chloroplast genomes, annotated from NCBI. All genomes stored as \textit{.fasta} files which have a collection of protein coding genes\cite{parra2007cegma,RDogma}(gene that produce proteins) with its coding sequences.
+As a preprocessing step to achieve the set of core genes, we need to analyse these genomes (using \textit{BioPython} package\cite{chapman2000biopython}), to extracting all information needed to find the core genes. The process starts by converting each genome from fasta format to GenVision\cite{geneVision} formats from DNASTAR. The outputs from this operation are lists of genes for each genome, their genes names and gene counts. In this stage, we accumulate some Gene duplications for each treated genome. In other words, duplication in gene name can comes from genes fragments, (e.g. gene fragments treated with NCBI), as long as chloroplast DNA sequences. To ensure that all the duplications are removed, each list of genes is translated into a set of genes. NCBI genome annotation produce genes except \textit{Ribosomal rRNA}.
-\begin{figure}[H]
-\caption{Extracting Core genome based on Gene Counts}
- \centering
- \includegraphics[width=0.7\textwidth]{NCBI_GeneName}
-\end{figure}
+\subsubsection{Genome annotation from Dogma}
+Dogma is an annotation tool developed in the university of Texas in 2004. Dogma is an abbreviation of (\textit{Dual Organellar GenoMe Annotator}) for plant chloroplast and animal mitochondrial genomes.
+It has its own database for translating the genome in all six reading frames and query the amino acid sequence database using Blast\cite{altschul1990basic}(i.e Blastx) with various parameters. Further more, identify protein coding genes in the input genome based on sequence similarity of genes in Dogma database. In addition, it can produce the \textit{Transfer RNAs (tRNA)}, and the \textit{Ribosomal RNAs (rRNA)} and verifies their start and end positions rather than NCBI annotation tool. There are no gene duplication with dogma after solving gene fragmentation. \\
+Genome Annotation with dogma can be the key difference of extracting core genes. The step of annotation divided into two tasks: First, It starts to annotate complete chloroplast genome (i.e \textit{Unannotate genome from NCBI} by using Dogma web tool. This process was done manually. The output from dogma is considered to be collection of coding genes file for each genome in the form of GeneVision file format.
+Where the second task is to solve gene fragments. Two methods used to solve genes duplication for extract core genes. First, for the method based on gene name, all the duplications are removed, where each list of genes is translated into a set of genes. Second, for the method of gene quality test, defragment process starts immediately to solve fragments of coding genes for each genome to avoid gene duplication. In each iteration, this process starts by taking one gene from gene list, search for gene duplication, if exists, look on the orientation of the fragment sequence: if it is positive, then appending fragment sequence to gene file. Otherwise, the process applies reverse complement operations on gene sequence and append it to gene file. Additional process applied to check start and stop codon and try to find appropriate start and end codon in case of missing. All genomes after this stage are fully annotated, their genes were de-fragmented, genes lists and counts were identified.\\
+
+\subsection{Core Genes Extraction}
+The goal of this step is to extract maximum core genes from sets of genes. The methodology of finding core genes is as follow: \\
+
+\subsubsection{Pre-Processing}
+We apply two pre-processing methods for organize and prepare genomes data, one method based on gene name and count, and the second method is based on sequence quality control test.\\
+In the first method, preparing chloroplasts genomes to extract core genes based on gene name and count starts after annotation process because genomes vary in genes counts and types according to the annotation used method. Then we store each genome in the database under genome name with the set of genes names. Genes counts can extracted simply by a specific length command. \textit{Intersection core matrix} will apply then to extract the core genes. The problem with this method is how we can quarantine that the gene predicted in core genes is the same gene in leaf genomes?. To answer this question, if the sequence of any gene in a genome annotated from dogma and NCBI are similar with respect to a threshold, we do not have any problem with this method. Otherwise, we have a problem, because we can not decide which sequence goes to a gene in core genes.
+The second pre-processing method state: we can predict the best annotated genome by merge the annotated genomes from NCBI and dogma based on the quality of genes names and sequences test. To generate all quality genes of each genome. the hypothesis state: Any gene will be in predicted genome if and only if the annotated genes between NCBI and Dogma pass a specific threshold of\textit{quality control test}. To accept the quality test, we applied Needle-man Wunch algorithm to compare two gene sequences with respect to pass a threshold. If the alignment score pass this threshold, then the gene will be in the predicted genome. Otherwise, the gene will be ignored. After predicting all genomes, \textit{Intersection core matrix} will apply on these new genomes to extract core genes. As shown 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, it is used to find the best similarity score between two gene sequences after applying operations like \textit{reverse, complement, and reverse complement}. The algorithm of geneChk is illustrated in Algorithm \ref{Alg3:genechk}.
+
+\begin{algorithm}[H]
+\caption{Find the Maximum similarity score between two sequences}
+\label{Alg3:genechk}
+\begin{algorithmic}
+\REQUIRE $gen1,gen2 \leftarrow \text{NCBI gene sequence, Dogma gene sequence}$
+\ENSURE $\text{Maximum similarity score}$
+\STATE $Score1 \leftarrow needle(gen1,gen2)$
+\STATE $Score2 \leftarrow needle(gen1,Reverse(gen2))$
+\STATE $Score3 \leftarrow needle(gen1,Complement(gen2))$
+\STATE $Score4 \leftarrow needle(gen1,Reverse(Complement(gen2)))$
+\IF {$max(Score1, Score2, Score3, Score4)==Score1$}
+ \RETURN $Score1$
+\ELSIF {$max(Score1, Score2, Score3, Score4)==Score2$}
+ \RETURN $Score2$
+\ELSIF {$max(Score1, Score2, Score3, Score4)==Score3$}
+ \RETURN $Score3$
+\ELSIF {$max(Score1, Score2, Score3, Score4)==Score4$}
+ \RETURN $Score4$
+\ENDIF
+\end{algorithmic}
+\end{algorithm}
+
+\subsubsection{Intersection Core Matrix (\textit{ICM})}
+
+The idea behind extracting core genes is to collect iteratively the maximum number of common genes between two genomes. To do so, the system builds an \textit{Intersection core matrix (ICM)}. ICM is a two dimensional symmetric matrix where each row and column represent one genome. Each position in ICM stores the \textit{intersection scores(IS)}. The Intersection Score is the cardinality number of a core genes which comes from intersecting one genome with other ones. Maximum cardinality results to select two genomes with their maximum core. Mathematically speaking, if we have an $n \times n$ matrix where $n=\text{number of genomes in local database}$, then lets consider:\\
-\textbf{Step II: Gene Intersection}\\
-The main objective of this step is try to find best core genes from sets of genes in the database. The idea for finding core genes is to collect in each iteration the maximum number of common genes. To do this, the system build an intersection core matrix(ICM). ICM here is a two dimensional symmetric matrix where each row and column represent the list of genomes in the local database. Each position in ICM stores the \textit{intersection scores}. Intersection Score(IS), is the score by intersect in each iteration two sets of genes for two different genomes in the database. Taking maximum score from each row and then taking the maximum of them will result to draw the two genomes with their maximum core. Then, the system remove these two genomes from ICM and add the core of them under a specific name to ICM for the calculation in next iteration. The core genes generated with its set of genes will store in a database for reused in the future. this process repeat until all genomes treated. If maximum intersection core(MIC) equal to 0, the system will avoid this intersection operation and ignore the genome that smash the maximum core genes.\\
-We observe that ICM will result to be very large because of the huge amount of data that it stores. In addition, this will results to be time and memory consuming for calculating the intersection scores by using just genes names. To increase the speed of calculations, we can calculate the upper triangle scores only and exclude diagonal scores. This will reduce whole processing time and memory to half. The time complexity for this process after enhancement changed from $O(n^2)$ to $O((n-1)\log{n})$.\\
-The Algorithm of construction the matrix and extracting maximum core genes where illustrated in Algorithm 1. The output from this step is list of core genes with their lengths to be drawn in a tree.
+\begin{equation}
+Score=\max_{i<j}\vert x_i \cap x_j\vert
+\label{Eq1}
+\end{equation}\\
+
+Where $x_i, x_j$ are elements in the matrix. The generation of a new core genes is depending on the cardinality value of intersection scores, we call it \textit{Score}:
+$$\text{New Core} = \begin{cases}
+\text{Ignored} & \text{if $\textit{Score}=0$;} \\
+\text{new Core id} & \text{if $\textit{Score}>0$.}
+\end{cases}$$
+
+if $\textit{Score}=0$ then we have \textit{disjoint relation} (i.e no common genes between two genomes). In this case the system ignores the genome that annul the core genes size. Otherwise, The system will removes these two genomes from ICM and add new core genomes with a \textit{coreID} of them to ICM for the calculation in next iteration. This process will reduce the size of ICM and repeat until all genomes are treated (i.e ICM has no more genomes).
+We observe that ICM is very large because of the amount of data that it stores. This results to be time and memory consuming for calculating the intersection scores. To increase the speed of calculations, it is sufficient to only calculate the upper triangle scores. The time complexity for this process after enhancement is thus $O(\frac{(n-1).n}{2})$. Algorithm \ref{Alg1:ICM} illustrates the construction of the ICM matrix and the extraction of the core genes where \textit{GenomeList}, represents the database where all genomes data are stored. At each iteration, it computes the maximum core genes with its two genomes parents.
\begin{algorithm}[H]
\caption{Extract Maximum Intersection Score}
+\label{Alg1:ICM}
\begin{algorithmic}
-\REQUIRE $L \leftarrow sets of genomes genes$
-\ENSURE $B1 \leftarrow Max core$
+\REQUIRE $L \leftarrow \text{genomes vectors}$
+\ENSURE $B1 \leftarrow Max core vector$
\FOR{$i \leftarrow 0:len(L)-1$}
\STATE $core1 \leftarrow set(GenomeList[L[i]])$
\STATE $score1 \leftarrow 0$
- \STATE $g1,g2 \leftarrow ''$
+ \STATE $g1,g2 \leftarrow$ " "
\FOR{$j \leftarrow i+1:len(L)$}
\STATE $core2 \leftarrow set(GenomeList[L[i]])$
\IF{$i < j$}
\ENDFOR
\STATE $B1[score1] \leftarrow (g1,g2)$
\ENDFOR
-\RETURN $B1$
+\RETURN $max(B1)$
\end{algorithmic}
\end{algorithm}
-In this algorithm, \textit{GenomeList} represents the database.
+\subsection{Features Visualization}
+The goal here is to visualize the results by building a tree of evolution. All Core genes generated with their genes are very important information in the tree, because they can be viewed as an ancestor information for two genomes or more. Further more, each node represents a genome or core as \textit{(Genes count:Family name, Scientific names, Accession number)}, Edges represent numbers of lost genes from genomes-core or core-core relationship. The number of lost genes here can represent an important factor for evolution, it represents how much lost of genes for the species in same or different families. By the principle of classification, small number of gene lost among species indicate that those species are close to each other and belong to same family, while big genes lost means that we have an evolutionary relationship between species from different families. To see the picture clearly, Phylogenetic tree is an evolutionary tree generated also by the system. Generating this tree is based on the distances among genes sequences. There are many resources to build such tree (for example: PHYML\cite{guindon2005phyml}, RAxML{\cite{stamatakis2008raxml,stamatakis2005raxml}, BioNJ , and TNT\cite{goloboff2008tnt}}. We consider to use RAxML\cite{stamatakis2008raxml,stamatakis2005raxml} because it is fast and accurate for build large trees for large count of genomes sequences. The procedure of constructing phylogenetic tree stated in the following steps:
+
+\begin{enumerate}
+\item Extract gene sequence for all gene in all core genes, store it in database.
+\item Use multiple alignment tool such as (****to be write after see christophe****) to align these sequences with each others.
+\item aligned genomes sequences then submitted to RAxML program to compute the distances and draw phylogenetic tree.
+\end{enumerate}
+
+\begin{figure}[H]
+ \centering
+ \includegraphics[width=0.7\textwidth]{Whole_system}
+ \caption{Total overview of the system pipeline}\label{wholesystem}
+\end{figure}
+
+\section{Implementation}
+We implemented four algorithms to extract maximum core genes from large amount of chloroplast genomes. Two algorithms used to extract core genes based on NCBI annotation, and the others based on dogma annotation tool. Evolutionary tree generated as a result from each method implementation. In this section, we will present the four methods, and how they can extract maximum core genes?, and how the developed code will generate the evolutionary tree.
-\textbf{Step III: Draw the Tree}\\
+\subsection{Extract Core Genes based on Gene Contents}
-The main drawback from this method is that we can not depending only on genes names because of three causes: first, the genome may have not totally named, so we will have some lost sequences. Second, may we have two genes sharing the same name, while their sequences are different. Third, we need to annotate 99 genomes.
+\subsubsection{Core Genes based on NCBI Annotation}
+The first idea to construct the core genome is based on the extraction of Genes names (as gene presence or absence). For instant, in this stage neither sequence comparison nor new annotation were made, we just want to extract all genes with counts stored in each chloroplast genome, then find the intersection core genes based on gene names. \\
+The pipeline of extracting core genes can summarize in the following steps according to pre-processing method used:\\
-\subsubsection{Extracting Core genome from NCBI gene contents}
+\begin{enumerate}
+\item We downloads already annotated chloroplast genomes in the form of fasta coding genes (i.e \textit{exons}).
+\item Extract genes names and apply to solve gene duplication using first method.
+\item Convert fasta file format to geneVision file format to generate ICM.
+\item Calculate ICM matrix to find maximum core \textit{Score}. New core genes for two genomes will generate and a specific \textit{CoreId} will assign to it. This process continue until no elements remain in the matrix.
+\item Evolutionary tree will take place by using all data generated from step 1 and 4. The tree will also display the amount of genes lost from each intersection iteration. A specific excel file will be generated that store all the data in local database.
+\end{enumerate}
+There main drawback with this method is genes orthography (e.g two different genes sequences with same gene name). In this case, Gene lost is considered by solving gene duplication based on first method to solve gene duplication.
-\subsection{Core genes from Dogma Annotation tool}
+\subsubsection{Core Genes based on Dogma Annotation}
+The main goal is to get as much as possible the core genes of maximum coding genes names. According to NCBI annotation problem based on \cite{Bakke2009}, annotation method like dogma can give us more reliable coding genes than NCBI. This is because NCBI annotation can carry some annotation and gene identification errors. The general overview of whole process of extraction illustrated in figure \ref{wholesystem}.
+extracting core genes based on genes names and counts summarized in the following steps:\\
+\begin{enumerate}
+\item We apply the genome annotation manually using Dogma annotation tool.
+\item Analysing genomes to store lists of code genes names (i.e \textit{exons}). solve gene fragments is done by using first method in solve gene fragments. The output from annotation process with dogma is genomes files in GenVision file format. Sets of genes were stored in the database.
+\item Generate ICM matrix to calculate maximum core genes.
+\item Draw the evolutionary tree by extracted all genes sequences from each core. Then applying multiple alignment process on the sequences to calculate the distance among cores to draw a phylogenetic tree.
-\subsubsection{Core genes based on Genes names and count}
+\end{enumerate}
-\begin{figure}[H]
-\caption{Extracting Core genome based on Gene Name}
- \centering
- \includegraphics[width=0.7\textwidth]{Dogma_GeneName}
-\end{figure}
+The main drawback from the method of extracting core genes based on gene names and counts is that we can not depending only on genes names because of three causes: first, the genome may have not totally named (This can be found in early versions of NCBI genomes), so we will have some lost sequences. Second, we may have two genes sharing the same name, while their sequences are different. Third, we need to annotate all the genomes.
-\subsubsection{Core genome from Dogma gene contents}
+\subsection{Extract Core Genes based on Gene Quality Control}
+The main idea from this method is to focus on genes quality to predict maximum core genes. By comparing only genes names from one annotation tool is not enough. The question here, does the predicted gene from NCBI is the same gene predicted by Dogma based on gene name and gene sequence?. If yes, then we can predict new quality genomes based on quality control test with a specific threshold. Predicted Genomes comes from merging two annotation techniques. While if no, we can not depending neither on NCBI nor Dogma because of annotation error. Core genes can by predicted by using one of the
-\begin{figure}[H]
-\caption{Extracting Core genome based on Gene Name}
- \centering
- \includegraphics[width=0.7\textwidth]{Dogma_GeneContent}
-\end{figure}
+\subsubsection{Core genes based on NCBI and Dogma Annotation}
+This method summarized in the following steps:\\
+
+\begin{enumerate}
+\item Retrieve the annotation of all genomes from NCBI and Dogma: in this step, we apply the annotation of all chloroplast genomes in the database using NCBI annotation and Dogma annotation tool.
+\item Convert NCBI genomes to GeneVision file format, then apply the second method of gene defragmentation methods for NCBI and dogma genomes.
+\item Predict quality genomes: the process is to pick a genome annotation from two sources, extracting all common genes based on genes names, then applying Needle-man wunch algorithm to align the two sequences based on a threshold equal to 65\%. If the alignment score pass the threshold, then this gene will removed from the competition and store it in quality genome by saving its name with the largest gene sequence with respect to start and end codons. All quality genomes will store in the form of GenVision file format.
+\item Extract Core genes: from the above two steps, we will have new genomes with quality genes, ofcourse, we have some genes lost here, because dogma produced tRNA and rRNA genes while NCBI did not generate rRNA genes and vise-versa. Build ICM to extract core genes will be sufficient because we already check genes sequences.
+\item Display tree: An evolution tree then will be display based on the intersections of quality genomes.
+\end{enumerate}
