-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.\\
-\begin{figure}[H]
-\caption{A general overview of the system}
- \centering
- \includegraphics[width=0.5\textwidth]{generalView}
-\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.
+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.
-\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]
+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=0.7\textwidth]{image2}
+ \includegraphics[width=0.75\textwidth]{generalView}
+\caption{A general overview of the annotation-based approach}\label{Fig1}
\end{figure}
-\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.
+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. To obtain relevant annotated
+genomes, two annotation techniques from NCBI and Dogma are used. 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.
-\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.\\
+\input{population_Table}
+
+\subsection{Genome annotation techniques}
-\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.
+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.
-\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 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.
-\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.
+\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. Another difference is also that
+there is no gene duplication with Dogma after solving gene
+fragmentation. 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 use 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, then we do not have any problem with this
+method. When the sequences are not similar we have a problem, because
+we cannot decide which sequence belongs to a gene in core genes.
+
+The second method is based on the underlying idea: we can 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: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 $B1$
+\RETURN $max(B1)$
\end{algorithmic}
\end{algorithm}
-In this algorithm, \textit{GenomeList} represents the database.\\
+\subsection{Features visualization}
-\textbf{Step III: Drawing the Tree}\\
-The main objective here is to the results for visualizing a tree of evolution. We use here a directed graph from Dot graph package\cite{gansner2002drawing} from Graphviz library. The system produce this tree automatically by using all information available in a database. Core genes generated with their genes can be very important information, because they represent also the ancestor information for two genomes. In this tree, each node represent genome or core genes as \textit{(Genes count:Family name,Scientific name,Accession number)}, Edges here represent the number of lost genes from genomes-core or core-core intersection. 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 genes lost among species can say that these species are closely together and belongs 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}\cite{stamatakis2005raxml}, BioNJ , and TNT\cite{goloboff2008tnt}}. We consider to use RAxML\cite{stamatakis2008raxml}\cite{stamatakis2005raxml} to generate this tree.
+The goal is to visualize results by building a tree of evolution. 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
+the evolution: how many genes were lost between two species whether
+they belong to the same family or not. By the principle of
+classification, a small number of genes lost among species indicates
+that those species are close to each other and belong to same family,
+while a large lost means that we have an evolutionary relationship
+between species from different families. 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}
+\begin{figure}[H]
+ \centering \includegraphics[width=0.75\textwidth]{Whole_system} \caption{Overview
+ of the pipeline}\label{wholesystem}
+\end{figure}
+\section{Implementation}
-\subsection{Core genes from Dogma Annotation tool}
+The different algorithms have been implemented using Python version
+2.7, on a laptop running Ubuntu~12.04~LTS. More precisely, the
+computer is a Dell Latitude laptop - model E6430 with 6~GiB memory and
+a quad-core Intel core~i5~processor with an operating frequency of
+2.5~GHz. Many python packages such as os, Biopython, memory\_profile,
+re, numpy, time, shutil, and xlsxwriter were used to extract core
+genes from large amount of chloroplast genomes.
+\begin{center}
+\begin{table}[b]
+\caption{Type of annotation, execution time, and core genes
+for each method}\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 & $\surd$ & - & - & $\surd$ & ? & - & ? & - & 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}
+
+\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 {\it Annotation} columns
+represent the algorithm used to annotate chloroplast genomes, the {\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} to extract core genes
+from large chloroplast genome. Only the gene quality method requires
+several days of computation (about 3-4 days) for sequence comparisons,
+once the quality genomes are construced it takes just 1.29~minutes to
+extract core gene. Thanks to this low execution times we can 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. {\it Bad
+genomes} gives the number of genomes that destroy core genes due to
+low number of gene intersection. \textit{NC\_012568.1 Micromonas
+pusilla} is the only genome which destroyed the core genome with NCBI
+annotations for both gene features and gene quality methods.
+
+The second important factor is the amount of memory being used by each
+methodology. Table \ref{mem} shows the memory usage of each
+method. We used a package from PyPI~(\textit{the Python Package
+Index}) named \textit{Memory\_profile} (located at~{\tt
+https://pypi.python.org/pypi}) to extract all the values in
+table~\ref{mem}. 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 used by the third method also
+depends on the size of each genome.
+
+\begin{center}
+\begin{table}[H]
+\caption{Memory usages in (MB) for each methodology}\label{mem}
+{\scriptsize
+\begin{tabular}{p{2.5cm}p{1.5cm}p{1cm}p{1cm}p{1cm}p{1cm}p{1cm}p{1cm}}
+\hline\hline
+Method& & Load Gen. & Conv. gV & Read gV & ICM & Core tree & Core Seq. \\
+\hline
+Gene prediction & ~ & ~ & ~ & ~ & ~ & ~ & ~\\
+\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}
+\end{center}
-\subsubsection{Core genes based on Genes names and count}
-\begin{figure}[H]
-\caption{Extracting Core genome based on Gene Name}
- \centering
- \includegraphics[width=0.7\textwidth]{Dogma_GeneName}
-\end{figure}
-\subsubsection{Core genome from Dogma gene contents}
-\begin{figure}[H]
-\caption{Extracting Core genome based on Gene Name}
- \centering
- \includegraphics[width=0.7\textwidth]{Dogma_GeneContent}
-\end{figure}
