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-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 also 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 \ref{Fig1}.\\
 
-\begin{figure}[H]
-\caption{A general overview of the system}
-  \centering
-    \includegraphics[width=0.5\textwidth]{generalView}
-	\label{Fig1}
-\end{figure}
+These  last years  the cost  of  sequencing genomes  has been  greatly
+reduced,  and thus  more and  more genomes  are  sequenced.  Therefore
+automatic annotation tools are required to deal with this continuously
+increasing amount of genomical data. Moreover, a reliable and accurate
+genome  annotation  process  is  needed  in order  to  provide  strong
+indicators for the study of life\cite{Eisen2007}.
 
-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.
-
-\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 \ref{Tab1}.\pagebreak
-
-\footnotesize 
-\setlength\LTleft{-30pt}            
-\setlength\LTright{-30pt}           
-\begin{longtable}{@{\extracolsep{\fill}}llllllllll@{}}
-
-\caption[NCBI Genomes Families]{List of family groups of Chloroplast Genomes from NCBI\label{Tab1}}\\
-	% Heading
-	\hline\hline
-	{\textbf{Category}} & {\textbf{Counts}} & {\textbf{Accession No}} & {\textbf{Scientific Name}} \\ 
-	\hline
-	%Entering First line
-	  & & NC\_001713.1 & Odontella sinensis \\ 
-	  & & NC\_008588.1 & Phaeodactylum tricornutum \\ 
-	  & & NC\_010772.1 & Heterosigma akashiwo \\ 
-	  & & NC\_011600.1 & Vaucheria litorea \\ 
-	  & & NC\_012903.1 & Aureoumbra lagunensis \\ 
-	  Algues Brunes & 11 & NC\_014808.1 & Thalassiosira oceanica \\ 
-	  & & NC\_015403.1 & Fistulifera sp \\ 
-	  & & NC\_016731.1 & Synedra acus \\ 
-	  & & NC\_016735.1 & Fucus vesiculosus \\ 
-	  & & NC\_018523.1 & Saccharina japonica \\ 
-	  & & NC\_020014.1 & Nannochloropsis gadtina \\ [1ex]
-	%Entering second group
-	  & & NC\_000925.1 & Porphyra purpurea \\ 
-	  Algues Rouges & 3 & NC\_001840.1 & Cyanidium caldarium \\ 
-	  & & NC\_006137.1 & Gracilaria tenuistipitata \\ [1ex]
-	 %Entering third group
-	  & & NC\_000927.1 & Nephroselmis olivacea \\ 
-	  & & NC\_002186.1 & Mesotigma viride \\ 
-	  & & NC\_005353.1 & Chlamydomonas reinhardtii \\ 
-	  & & NC\_008097.1 & Chara vulgaris \\ 
-	  & & NC\_008099.1 & Oltmannsiellopsis viridis \\ 
-	  & & NC\_008114.1 & Pseudoclonium akinetum \\ 
-	  & & NC\_008289.1 & Ostreococcus tauri \\ 
-	  & & NC\_008372.1 & Stigeoclonium helveticum \\ 
-	  Algues Vertes & 17 & NC\_008822.1 & Chlorokybus atmophyticus \\ 
-	  & & NC\_011031.1 & Oedogonium cardiacum \\ 
-	  & & NC\_012097.1 & Pycnococcus provaseolii \\ 
-	  & & NC\_012099.1 & Pyramimonas parkeae \\ 
-	  & & NC\_012568.1 & Micromonas pusilla \\ 
-	  & & NC\_014346.1 & Floydiella terrestris \\ 
-	  & & NC\_015645.1 & Schizomeris leibleinii \\ 
-	  & & NC\_016732.1 & Dunaliella salina \\ 
-	  & & NC\_016733.1 & Pedinomonas minor \\ [1ex] 
-	 %Entering fourth group
-	  & & NC\_001319.1 & Marchantia polymorpha \\ 
-	  Bryophytes & 3 & NC\_004543.1 & Anthoceros formosae \\ 
-	  & & NC\_005087.1 & Physcomitrella patens \\ [1ex]
-	 %Entering fifth group
-	  & & NC\_014267.1 & Kryptoperidinium foliaceum \\ 
-	  Dinoflagelles & 2
-	  	& NC\_014287.1 & Durinskia baltica \\ [1ex]
-	 %Entering sixth group
-	  & & NC\_001603.2 & Euglena gracilis \\ 
-	  Euglenes & 2 & NC\_020018.1 & Monomorphina aenigmatica \\ [1ex]
-	 %Entering seventh group
-	  & & NC\_003386.1 & Psilotum nudum \\ 
-	  & & NC\_008829.1 & Angiopteris evecta \\
-	  Filicophytes & 5 & NC\_014348.1 & Pteridium aquilinum \\ 
-	  & & NC\_014699.1 & Equisetum arvense \\ 
-	  & & NC\_017006.1 & Mankyua chejuensis \\ [1ex]
-	  % Entering eighth group
-	  & & NC\_001568.1 & Epifagus virginiana \\ 
-	  & & NC\_001666.2 & Zea Mays \\ 
-	  & & NC\_005086.1 & Amborella trichopoda \\ 
-	  & & NC\_006050.1 & Nymphaea alba \\ 
-	  & & NC\_006290.1 & Panax ginseng \\ 
-	  & & NC\_007578.1 & Lactuca sativa \\ 
-	  & & NC\_007957.1 & vitis vinifera \\ 
-	  & & NC\_007977.1 & Helianthus annuus \\ 
-	  & & NC\_008325.1 & Daucus carota \\ 
-	  & & NC\_008336.1 & Nandina domestica \\ 
-	  & & NC\_008359.1 & Morus indica \\ 
-	  & & NC\_008407.1 & Jasminum nudiflorum \\ 
-	  & & NC\_008456.1 & Drimys granadensis \\ 
-	  & & NC\_008457.1 & Piper cenocladum \\ 
-	  & & NC\_009601.1 & Dioscorea elephantipes \\ 
-	  & & NC\_009765.1 & Cuscuta gronovii \\ 
-	  & & NC\_009808.1 & Ipomea purpurea \\ 
-	 Angiospermes & 45 & NC\_010361.1 & Oenothera biennis \\ 
-	  & & NC\_010433.1 & Manihot esculenta \\ 
-	  & & NC\_010442.1 & Trachelium caeruleum \\ 
-	  & & NC\_013707.2 & Olea europea \\ 
-	  & & NC\_013823.1 & Typha latifolia \\ 
-	  & & NC\_014570.1 & Eucalyptus \\ 
-	  & & NC\_014674.1 & Castanea mollissima \\ 
-	  & & NC\_014676.2 & Theobroma cacao \\ 
-	  & & NC\_015830.1 & Bambusa emeiensis \\ 
-	  & & NC\_015899.1 & Wolffia australiana \\ 
-	  & & NC\_016433.2 & Sesamum indicum \\ 
-	  & & NC\_016468.1 & Boea hygrometrica \\ 
-	  & & NC\_016670.1 & Gossypium darwinii \\ 
-	  & & NC\_016727.1 & Silene vulgaris \\ 
-	  & & NC\_016734.1 & Brassica napus \\ 
-	  & & NC\_016736.1 & Ricinus communis \\ 
-	  & & NC\_016753.1 & Colocasia esculenta \\ 
-	  & & NC\_017609.1 & Phalaenopsis equestris \\ 
-	  & & NC\_018357.1 & Magnolia denudata \\ 
-	  & & NC\_019601.1 & Fragaria chiloensis \\ 
-	  & & NC\_008796.1 & Ranunculus macranthus \\ 
-	  & & NC\_013991.2 & Phoenix dactylifera \\ 
-	  & & NC\_016068.1 & Nicotiana undulata \\ [1ex]
-	  %Entering ninth group
-	  & & NC\_009618.1 & Cycas taitungensis \\ 
-	  & & NC\_011942.1 & Gnetum parvifolium \\ 
-	  & & NC\_016058.1 & Larix decidua \\ 
-	  Gymnosperms & 7 & NC\_016063.1 & Cephalotaxus wilsoniana \\ 
-	  & & NC\_016065.1 & Taiwania cryptomerioides \\ 
-	  & & NC\_016069.1 & Picea morrisonicola \\ 
-	  & & NC\_016986.1 & Gingko biloba \\ [1ex]
-	  %Entering tenth group
-	  Haptophytes & 1 & NC\_007288.1 & Emiliana huxleyi\\ [1ex]
-	  %Entering eleventh group
-	  Lycophytes & 2 & NC\_014675.1 & Isoetes flaccida \\
-	  & & NC\_006861.1 & Huperzia lucidula \\
-	\hline
-\end{longtable}
-
-\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.
-
-\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.\\
-
-\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 of genomes from NCBI stored as \textit{.fasta} files that include a collection of Protein coding genes\cite{parra2007cegma,RDogma}(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 using \textit{BioPython} package\cite{chapman2000biopython}, and extracting all information needed to find the core genes. The process starts by converting each genome in fasta format to GenVision\cite{geneVision} format from DNASTAR, and 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. We defines \textit{Identical state} to be 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. This state 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 form one gene. 
-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 $set(G)=\{g_1,g_2,g_3,g_4\}$, and $|G|=4$ where $|G|$ is the cardinality number of the set $G$ which represent the number of genes in the set. 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. In our method, this can generate the problem of gene lost 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 \ref{Fig2}.
+Various  annotation   tools  (\emph{i.e.},  cost-effective  sequencing
+methods\cite{Bakke2009}) producing genomic  annotations at many levels
+of detail  have been designed  by different annotation  centers. Among
+the major annotation  centers we can notice NCBI\cite{Sayers01012011},
+Dogma       \cite{RDogma},       cpBase      \cite{de2002comparative},
+CpGAVAS                   \cite{liu2012cpgavas},                   and
+CEGMA\cite{parra2007cegma}. Usually, previous  studies used one out of
+three methods  for finding  genes in annoted  genomes using  data from
+these  centers: \textit{alignment-based},  \textit{composition based},
+or a  combination of both~\cite{parra2007cegma}.   The alignment-based
+method  is used  when trying  to predict  a coding  gene (\emph{i.e.}.
+genes that produce proteins) by aligning a genomic DNA sequence with a
+cDNA  sequence  coding  an homologous  protein  \cite{parra2007cegma}.
+This approach is  also used in GeneWise\cite{birney2004genewise}.  The
+alternative   method,   the    composition-based   one   (also   known
+as  \textit{ab initio})  is based  on  a probabilistic  model of  gene
+structure  to  find genes  according  to  the  gene value  probability
+(GeneID \cite{parra2000geneid}).  Such  annotated genomic data will be
+used to overcome  the limitation of the first  method described in the
+previous section.   In fact, the  second method we propose  finds core
+genes  from  large  amount  of  chloroplast  genomes  through  genomic
+features extraction.
 
-\begin{figure}[H]
+Figure~\ref{Fig1} presents an overview  of the entire method pipeline.
+More    precisely,    the   second    method    consists   of    three
+stages:   \textit{Genome    annotation},   \textit{Core   extraction},
+and    \textit{Features    Visualization}    which   highlights    the
+relationships.  To  understand the  whole core extraction  process, we
+describe briefly each  stage below. More details will  be given in the
+coming subsections.   The method uses as starting  point some sequence
+database  chosen  among   the  many  international  databases  storing
+nucleotide sequences, like  the GenBank at NBCI \cite{Sayers01012011},
+the    \textit{EMBL-Bank}     \cite{apweiler1985swiss}    in    Europe
+or   \textit{DDBJ}   \cite{sugawara2008ddbj}   in  Japan.    Different
+biological tools can analyze  and annotate genomes by interacting with
+these databases to  align and extract sequences to  predict genes. The
+database in  our method must be  taken from any  confident data source
+that stores annotated and/or unannotated chloroplast genomes.  We have
+considered the GenBank-NCBI \cite{Sayers01012011} database as sequence
+database:  99~genomes of chloroplasts  were retrieved.   These genomes
+lie in  the eleven type  of chloroplast families and  Table \ref{Tab2}
+summarizes their distribution in our dataset.
+
+\begin{figure}[h]  
   \centering
-    \includegraphics[width=0.9\textwidth]{NCBI_GeneName}
-    
-  \caption{Extracting Core genes based on NCBI Gene name and Counts}
-  \label{Fig2}
+    \includegraphics[width=0.75\textwidth]{generalView}
+\caption{A general overview of the annotation-based approach}\label{Fig1}
 \end{figure}
 
-\textbf{Step II: Gene Intersection}\\
-The goal of this step is trying to find maximum 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 \textit{Intersection core matrix(ICM)}. ICM here is a two dimensional symmetric matrix where each row and column represent a set of genes for one genome in the local database. Each position in ICM stores the \textit{intersection scores}. Intersection Score(IS), is the cardinality number of a core genes comes from intersecting in each iteration the set of genes for one genome with all other gene sets belong to the rest of genomes in the database. Taking maximum cardinality from each row and then taking the maximum of them will result to select the best two genomes with their maximum core. Mathematically speaking, if we have an $mxn$ matrix where $m,n=$number of genomes in database. lets consider $Z=max_{i<j}(\vert x_i \cap x_j\vert)$ where $x_i, x_j$ are sets of row and column elements in a matrix. if $Z=0$ then we have \textit{disjoint relation} (i.e no common genes between to genomes). In this case the system ignore the set of genes that smash the core genes. Otherwise, The system remove these two genomes from ICM and add new core with a coreID of them to ICM for the calculation in next iteration. The partial core genes generated with its set of genes will store in a database for reused for drawing the tree. this process repeat until all genomes treated.   
-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 that is why we write $max_{i<j}$. This will reduce whole processing time and memory to half. The time complexity for this process after  enhancement changed from $O(n^2)$ to $O((n-1)\log{n})$.\\
+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 Algorithm of construction the matrix and extracting maximum core genes where illustrated in Algorithm \ref{Alg1}. The output from this step is the maximum core genes with its genomes to draw it in a tree.  
+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
 
 \begin{algorithm}[H]
 \caption{Extract Maximum Intersection Score}
-\label{Alg1}
+\label{Alg1:ICM}
 \begin{algorithmic} 
-\REQUIRE $L \leftarrow sets of genomes genes$
-\ENSURE $B1 \leftarrow Max core$ 
-\FOR{$i \leftarrow 0:len(L)-1$}
+\REQUIRE $L \leftarrow \text{genomes sets}$
+\ENSURE $B1 \leftarrow \text{Max Core set}$ 
+\FOR{$i \leftarrow 1:len(L)-1$}
+        \STATE $score \leftarrow 0$
 	\STATE $core1 \leftarrow set(GenomeList[L[i]])$
-	\STATE $score1 \leftarrow 0$
-	\STATE $g1,g2 \leftarrow$ " "
+	\STATE $g1 \leftarrow L[i]$
 	\FOR{$j \leftarrow i+1:len(L)$}
-		\STATE $core2 \leftarrow set(GenomeList[L[i]])$
-		\IF{$i < j$}
-			\STATE $Core \leftarrow core1 \cap core2$
-			\IF{$len(Core) > score1$}
-				\STATE $g1 \leftarrow L[i]$
-				\STATE $g2 \leftarrow L[j]$
-				\STATE $Score \leftarrow len(Core)$
-			\ELSIF{$len(Core) == 0$}
-				\STATE $g1 \leftarrow L[i]$
-				\STATE $g2 \leftarrow L[j]$
-				\STATE $Score \leftarrow -1$
-			\ENDIF
-		\ENDIF
+	        \STATE $core2 \leftarrow set(GenomeList[L[j]])$
+		\STATE $core \leftarrow core1 \cap core2$
+		\IF{$len(core) > score$}
+                  \STATE $score \leftarrow len(core)$
+		  \STATE $g2 \leftarrow L[j]$
+                \ENDIF
 	\ENDFOR
-	\STATE $B1[score1] \leftarrow (g1,g2)$
+	\STATE $B1[score] \leftarrow (g1,g2)$
 \ENDFOR
 \RETURN $max(B1)$
 \end{algorithmic}
 \end{algorithm}
 
-\textit{GenomeList} represents the database.\\
+\subsection{Features visualization}
 
-\textbf{Step III: Drawing the Tree}\\
-The goal here is to visualizing the results by build a tree of evolution. The system can produce this tree automatically by using Dot graphs package\cite{gansner2002drawing} from Graphviz library and all information available in a database. Core genes generated with their genes can be very important information in the tree, because they can 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 be 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 related together and belong to same family, while big genes lost means that species is far to be in the same family. 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} to generate this tree.   
+The goal is to visualize results  by building an evolutionary tree. All
+core  genes generated  represent  an important information  in the  tree,
+because they  provide ancestor information of two  or more
+genomes. Each  node in the  tree represents one chloroplast  genome or
+one predicted core and labelled as \textit{(Genes count:Family name\_Scientific
+names\_Accession number)}. While an edge is labelled with the number of
+lost  genes from  a leaf  genome or  an intermediate  core  gene. Such
+numbers are  very interesting because  they give an  information about
+evolution:  how many genes  were lost between two  species whether
+they  belong  to  the  same  lineage  or not. To depict  the links between
+species   clearly,  we   built   a  phylogenetic   tree  showing   the
+relationships based on the distances among genes sequences. Many tools
+are    available   to    obtain    a   such    tree,   for    example:
+PHYML\cite{guindon2005phyml},
+RAxML{\cite{stamatakis2008raxml,stamatakis2005raxml},    BioNJ,    and
+TNT\cite{goloboff2008tnt}}.    In   this  work,   we   chose  to   use
+RAxML\cite{stamatakis2008raxml,stamatakis2005raxml}   because   it  is
+fast, accurate,  and can build large  trees when dealing  with a large
+number of genomic sequences.
 
-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 (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.
+The procedure used to built a phylogenetic tree is as follows:
+\begin{enumerate}
+\item For each gene in a core gene, extract its sequence and store it in the database.
+\item Use multiple alignment tools such as (****to be write after see christophe****) 
+to align these sequences with each others.
+\item Use an outer-group genome from cyanobacteria to calculate distances.
+\item Submit the resulting aligned sequences to RAxML program to compute 
+the distances and finally draw the phylogenetic tree.
+\end{enumerate} 
 
-\subsubsection{Extracting Core genome from NCBI gene contents}
-{to do later}
+\begin{figure}[H]
+  \centering \includegraphics[width=0.75\textwidth]{Whole_system} \caption{Overview
+  of the pipeline}\label{wholesystem}
+\end{figure}
 
-\subsection{Core genes from Dogma Annotation tool}
-In previous section, extracting core genes based on NCBI annotation caused some lost of genes due to annotation process. Annotation can play an important role for these losts, because it represents the first process of gene identification. Good annotation tool still be challenged subject. (Genis Parra in 2007) published a paper state that the subject of accurately genomic and/or gene annotation is still an open source problem, even in the best case scenario where any project has all the expert biologists resources to annotate gene structures, the catalogues of genes can still unclear and still less accurate than experts. Where \cite{Bakke2009} also state ("Errors in the annotations are routinely deposited in databases such as NCBI and used to validate subsequent annotation errors."). So, good core genes still needs good annotation tool. A lot of software todayâs were developed for extracted core genes for eukaryote and prokaryote organisms such as CEGMA\cite{parra2007cegma}, Coregenes 3.0\cite{zafar2002coregenes}, and Dogma\cite{RDogma}. The appropriate annotation tool for plant chloroplast and mitochondrial genomes is Dogma.
+\section{Implementation}
 
-\subsubsection{Why Dogma rather than NCBI annotation?}
-Dogma is an annotation tool developed in the university of Texas by \cite{RDogma} in 2004. Dogma is an abbreviation of \textit{Dual Organellar GenoMe Annotator}\cite{RDogma} for plant chloroplast and animal mitochondrial genomes.
-It has its own database for translated the genome in all six reading frames and query the amino acid sequence database using Blast\cite{altschul1990basic}(i.e Blastx) with various parameters, and to identify protein coding genes\cite{parra2007cegma,RDogma} in the input genome based on sequence similarity of genes in Dogma database. Further more, it can produce the \textit{Transfer RNAs (tRNA)}\cite{RDogma}, and the \textit{Ribosomal RNAs (rRNA)}\cite{RDogma} and verifying their start and end positions rather than NCBI annotation tool.
+All the different  algorithms have  been implemented using  Python on a personal computer running Ubuntu~12.04 with 6~GiB memory and
+a  quad-core Intel  core~i5~processor with  an operating  frequency of
+2.5~GHz. All the programs can be downloaded at \url{http://......} .
+genes  from large  amount of  chloroplast  genomes.
 
-\subsubsection{Core genes based on Dogma Genes names and counts}
-The main goal is to get as much as possible the core genes of maximum coding genes. According to \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 fiqure \ref{dog:Fig1}, the pipeline of extracting core genes can summarize in the following steps:\\
+\begin{center}
+\begin{table}[H]
+\caption{Type of annotation, execution time, and core genes.}\label{Etime}
+{\scriptsize
+\begin{tabular}{p{2cm}p{0.5cm}p{0.25cm}p{0.5cm}p{0.25cm}p{0.5cm}p{0.25cm}p{0.5cm}p{0.25cm}p{0.5cm}p{0.2cm}}
+\hline\hline
+ Method & \multicolumn{2}{c}{Annotation} & \multicolumn{2}{c}{Features} & \multicolumn{2}{c}{Exec. time (min.)} & \multicolumn{2}{c}{Core genes} & \multicolumn{2}{c}{Bad genomes} \\
+~ & N & D & Name & Seq & N & D & N & D & N & D \\
+\hline
+Gene prediction & $\surd$ & - & - & $\surd$ & 1.7 & - & ? & - & 0 & -\\[0.5ex]
+Gene Features & $\surd$ & $\surd$ & $\surd$ & - & 4.98 & 1.52 & 28 & 10 & 1 & 0\\[0.5ex]
+Gene Quality & $\surd$ & $\surd$ & $\surd$ & $\surd$ & \multicolumn{2}{c}{$\simeq$3 days + 1.29} & \multicolumn{2}{c}{4} & \multicolumn{2}{c}{1}\\[1ex]
+\hline
+\end{tabular}
+}
+\end{table}
+\end{center} 
 
-\begin{figure}[H]
-  \centering
-    \includegraphics[width=0.7\textwidth]{Dogma_GeneName}
-    \label{dog:Fig1}
-    \caption{Core genome based on Dogma Gene Name and count}
-\end{figure}
+\vspace{-1cm}
+
+Table~\ref{Etime}  presents  for  each  method  the  annotation  type,
+execution time,  and the  number of core  genes. We use  the following
+notations:  \textbf{N}  denotes NCBI,  while  \textbf{D} means  DOGMA,
+and \textbf{Seq}  is for sequence. The first two {\it Annotation} columns
+represent the algorithm used to annotate chloroplast genomes. The next two ones {\it
+Features} columns mean  the kind  of gene feature used to extract core
+genes: gene name, gene sequence, or  both of them. It can be seen that
+almost all methods need low {\it Execution time} expended in minutes to extract core genes
+from the large set of chloroplast genomes. Only the gene quality method requires
+several days of computation (about 3-4 days) for sequence comparisons. However,
+once the quality genomes are well constructed, it only takes 1.29~minutes to
+extract core gene. Thanks to this low execution times that gave us a privilege to use these
+methods to extract core genes  on a personal computer rather than main
+frames or parallel computers. The lowest execution time: 1.52~minutes,
+is obtained with the second method using Dogma annotations. The number
+of {\it  Core genes} represents the  amount of genes in  the last core
+genome. The main goal is to  find the maximum core genes that simulate
+biological background of chloroplasts. With  NCBI we have 28 genes for
+96   genomes,   instead   of    10   genes   for   97   genomes   with
+Dogma. Unfortunately, the biological distribution of genomes with NCBI
+in core tree do not  reflect good biological perspective, whereas with
+DOGMA the  distribution of genomes is biologically  relevant. Some a few genomes maybe destroying core genes due to
+low  number  of  gene  intersection. More precisely, \textit{NC\_012568.1  Micromonas pusilla} is the only genome who destroyes the core genome with NCBI
+annotations for both gene features and gene quality methods.
+
+The second important factor is the amount of memory nessecary in each
+methodology.   Table   \ref{mem}  shows  the  memory   usage  of  each
+method. In this table, the values are  presented in megabyte
+unit and \textit{gV} means  genevision~file~format. We can notice that
+the level  of memory which is  used is relatively low  for all methods
+and is available  on any personal computer. The  different values also
+show that the gene features  method based on Dogma annotations has the
+more   reasonable   memory   usage,   except  when   extracting   core
+sequences. The third method gives the lowest values if we already have
+the   quality   genomes,   otherwise   it  will   consume   far   more
+memory. Moreover, the  amount of memory, which is used by the third method also
+depends on the size of each genome.
+
+
+\begin{table}[H]
+\centering
+\caption{Memory usages in (MB) for each methodology}\label{mem}
+\tabcolsep=0.11cm
+{\scriptsize
+\begin{tabular}{p{2.5cm}@{\hskip 0.1mm}p{1.5cm}@{\hskip 0.1mm}p{1cm}@{\hskip 0.1mm}p{1cm}@{\hskip 0.1mm}p{1cm}@{\hskip 0.1mm}p{1cm}@{\hskip 0.1mm}p{1cm}@{\hskip 0.1mm}p{1cm}}
+\hline\hline
+Method& & Load Gen. & Conv. gV & Read gV & ICM & Core tree & Core Seq. \\
+\hline
+Gene prediction & NCBI & 108 & - & - & - & - & -\\
+\multirow{2}{*}{Gene Features} & NCBI & 15.4 & 18.9 & 17.5 & 18 & 18 & 28.1\\
+              & DOGMA& 15.3 & 15.3 & 16.8 & 17.8 & 17.9 & 31.2\\
+Gene Quality  & ~ & 15.3 & $\le$3G & 16.1 & 17 & 17.1 & 24.4\\ 
+\hline
+\end{tabular}
+}
+\end{table}
+ 
 
-\textbf{Step I: Pre-processing}\\
-Pre-processing step represents the key difference between methods of extracting core genes. In figure \ref{dog:Fig1}, The pre-processing step can be divided into two tasks: First, It starts to annotate our genome samples in the form of complete genomes (i.e \textit{Unannotated genomes} from NCBI by using Dogma web tool. The whole annotation process by using dogma website is done manually. The output from the annotation process is considered to be a collection of coding genes file for each genome in the form of GeneVision\cite{geneVision} file format.\\
-Where the second task is to solve gene fragments. Defragment process starts here to solve fragments of coding genes for each genome, this process can avoid gene duplication. All genomes now are fully annotated, their genes were de-fragmented, and genes list and counts identified. These information stored in local database.\\
 
-From these two tasks, we can obtain clearly one copy of coding genes. To ensure that genes produces from dogma annotation process is same as the genes in NCBI. We apply separately a quality check process that align the same gene from dogma and NCBI with respect to a specific threshold.\\
 
-\textbf{Step II: Extraction Core genes}\\
-Extracting core genes will use the same process presented in the section of extracting core genes based on NCBI genes. ICM matrix will be considered by calculating the upper triangular cardinality cores to save time and to find the maximum length of core genes from each iteration see algorithm \ref{Alg1}. From each iteration, two genomes are considered to draw with their maximum cardinality core genes until no genome remain in the database. The key point here is that the intersection genome that smash the core genes in each iteration will be ignored from this competition.\\
 
-\textbf{Step III: Draw the tree}
-To build the tree of evolution for genomes. The algorithm is considered to take from the data base the first coreID generated from step two and   draw sequentially all the genomes that create this core. Sometimes, we have a core genome that intersect with another one. This tree also represented as a set of nodes which represent genome names and a set of edges, which represent the number of gene lost from each genome. Phylogenetic tree also considered here by using RAxML{\cite{stamatakis2008raxml,stamatakis2005raxml} based on calculating the distances among core genes in the database. 
 
-\subsubsection{Core genome from Dogma gene contents}
-[To do Later]
-\begin{figure}[H]
-  \centering
-    \includegraphics[width=0.7\textwidth]{Dogma_GeneContent}
-    \label{dog:Fig2}
-    \caption{Core genes based on the comparison of Dogma Genes Sequences}
-\end{figure}
\ No newline at end of file
