A Local database attache with each pipe stage to store all information 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 8 genomes considered to be not good. The remain 99 genomes lies in the 11 types of chloroplast families, as shown in Table \ref{Tab1}. The list of distribution of genomes is illstrated in detail in Table \ref{Tab2}.
+In this research, we retrieved 107 genomes of Chloroplasts from NCBI. 99 genomes of then is considered to working with. These genomes lies in the 11 types of chloroplast families, as shown in Table \ref{Tab1}. The list of distribution of genomes is illstrated in detail in Table \ref{Tab2}.
\begin{table}[H]
\caption{distribution on Chloroplast Families}\label{Tab1}
\input{population_Table}
\subsection{Genome Annotation Techniques}
-The second stage in system pipeline is genome annotation. Many annotation techniques were developed for annotate chloroplast genomes but they vary in the number and type of predicting genes (i.e the ability to predict genes and \textit{Transfere RNA (tRNA)} and \textit{Ribosomal RNA (rRNA)} genes). Two annotation techniques from NCBI and Dogma are considered to analyse chloroplast genomes to examin the accuricy of predicted coding genes. Figure \ref{NCBI_annotation}, illstrate two annotation technique.\\
+Genome annotation is considered the second stage in model pipline. Many annotation techniques were developed for 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: Transfere 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. Figure \ref{NCBI_annotation}, illstrate two annotation technique.\\
\begin{figure}[H]
\centering
\caption{Genome annotation using either NCBI or Dogma}\label{NCBI_annotation}
\end{figure}
-With each annotation model, we provide a quality check class for the flow of chloroplast genomes. This class has an access to NCBI taxonomy database based on genome accession number to retreive information for the genome. These information contains \textit{[Scientific name, lineage, Division, taxonomy ID, parentID, and Accession No]}. Examin each genome with this class (i.e based on some parameters), can ignore some genomes from this competition that not match a specific control condition.
+With each annotation model, we provide a quality check class for the flow of chloroplast genomes. This class has a direct access to NCBI taxonomy database based on genome accession number to retreive information for the genome. These information contains \textit{[Scientific name, lineage, Division, taxonomy ID, parentID, and Accession No]}. Examining each genome with this class (i.e based on some parameters), can ignore some genomes from this competition that not match a specific control condition.
\subsubsection{genome annotation from NCBI}
The objective from this step is to organize, solve genes duplications, and generate sets of genes from each genome. The input to the system is our list of chloroplast genomes, annotated from NCBI\cite{Sayers01012011}. All genomes stored as \textit{.fasta} files include collection of Protein coding genes\cite{parra2007cegma,RDogma}(gene that produce proteins) with its coding sequences.
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. There are no gene duplication with dogma after solving gene fragmentation. \\
Genome Anntation with dogma can be the key difference of extracting core genes. In figure \ref{dog:Annotation}, The step of annotation divided into two tasks: First, It starts to annotate complete choloroplast genomes (i.e \textit{Unannotated genomes} from NCBI by using Dogma web tool. The whole annotation 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\cite{geneVision} file format.\\
-Where the second task is to solve gene fragments. Defragment process starts immediately after the first task to solve fragments of coding genes for each genome to avoid gene duplication. All genomes after this stage are fully annotated, their genes were de-fragmented, genes lists and counts were identified. These information stored in local database.\\
+Where the second task is to solve gene fragments. Defragment process starts immediately after the first task to solve fragments of coding genes for each genome to avoid gene duplication. This process will looks on fragement orientation, if it is negative, then the process apply reverse complement operations on gene sequence. All genomes after this stage are fully annotated, their genes were de-fragmented, genes lists and counts were identified. These information stored in local database.\\
\begin{figure}[H]
\centering
\includegraphics[width=0.7\textwidth]{Dogma_GeneName}
\end{cases}$$\\
if $Score=0$ then we have \textit{disjoint relation} (i.e no common genes between two genomes). In this case the system ignore the vector that smash the core genes. Otherwise, The system will remove these two vectors from ICM and add new core vector with a \textit{coreID} of them to ICM for the calculation in next iteration. The partial core vectors generated with its values will store in the local database for reused to draw the tree. this process repeat until all vectors 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. This will reduce whole processing time and memory to half. The time complexity for this process after enhancement changed from $O(n^2-n)$ to $O(\frac{(n-1).n}{2})$. The Algorithm of construction the vector matrix and extracting the vector of maximum core genes where illustrated in Algorithm \ref{Alg1}. The output from this step is the maximum core vector with its two vectors to draw it in a tree.\\
+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 $O(n^2-n)$ to $O(\frac{(n-1).n}{2})$. The Algorithm of construction the vector matrix and extracting the vector of maximum core genes where illustrated in Algorithm \ref{Alg1:ICM}. The output from this step is the maximum core vector with its two vectors to draw it in a tree.\\
\begin{algorithm}[H]
\caption{Extract Maximum Intersection Score}
-\label{Alg1}
+\label{Alg1:ICM}
\begin{algorithmic}
\REQUIRE $L \leftarrow \text{genomes vectors}$
\ENSURE $B1 \leftarrow Max core vector$
\end{algorithm}
\textit{GenomeList} represents the local database.\\
-In second Method, due to the number of annotated genomes, annotate each genome can be very exhausted task specially with Dogma, because dogma offer a web tool for annotation, so that, each genome must annotate using this web tool. This operation need to do manually. We prefer to recover this problem by choosing one reference chloroplast and querying each reference gene by using \textit{Blastn} to examin its existance in remaining unannotated genomes in blast database. collect all match genomes from each gene hits, to satisfy the hypothesis "the gene who exists in maximum number of genomes also exist in a core genes". In addition, we can also extract the maximum core genes by examine how many genes present with each genome?.\\
+In second Method, due to the number of annotated genomes, annotate each genome can be very exhausted task specially with Dogma, because dogma offer a web tool for annotation, so that, each genome must annotate using this web tool. This operation need to do manually. We prefer to recover this problem by choosing one reference chloroplast and querying each reference gene by using \textit{Blastn} to examin its existance in remaining unannotated genomes in blast database. collect all match genomes from each gene hits, to satisfy the hypothesis "the gene who exists in maximum number of genomes also exist in a core genes". In addition, we can also extract the maximum core genes by examine how many genes present with each genome?. Algorithm \ref{Alg2:secondM}, state the general algorithm for second method. \\
+
+\begin{algorithm}[H]
+\caption{Extract Maximum Core genes based on Blast}
+\label{Alg2:secondM}
+\begin{algorithmic}
+\REQUIRE $Ref\_Genome \leftarrow \text{Accession No}$
+\ENSURE $Core \leftarrow \text{Genes in each genome}$
+\FOR{$i \leftarrow Ref\_Genome$}
+ \STATE $G\_list=[ ]$
+ \STATE $File \leftarrow Blastn(i)$
+ \STATE $G\_list \leftarrow File[\text{Genomes names}]$
+ \STATE $Core \leftarrow [Accession\_No:G\_list]$
+\ENDFOR
+\RETURN $Core$
+\end{algorithmic}
+\end{algorithm}
The hypothesis in last 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. 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, else the gene will be ignored. After predicting all genomes, one of previous two methods can be applied to extract core genes.
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.
\subsection{Extract Core Genes based on Genes Sequences}
+We discussed before on the hypothesis of the second method. In this section, we will implement this hypothesis by using ncbi-Blast alignment tool. Implementation of this method is dividing into two parts: \textit{Core genes from NCBI Annotation} and \textit{Core Genes from Dogma Annotation}. For instance, for the two parts, selecting a reference genome can be a key difference among predicting Core genes. After choosing a reference genome, Local blast database will then created to store the rest of Un-annotated chloroplast genomes. \\
+
+We will present the algorithm in the following steps:
-\subsubsection{Core Genes from NCBI Annotation}
+\begin{enumerate}
+\item Select a reference genome: we need to select good reference genome from our population, To do so, we can choose \textit{Lycopersicon esculentum cultivar LA3023 chloroplast NC\_007898.3} to be the reference genome if we consider the version of annotation, or \textit{Zea Mays NC\_001666.2} if we consider the largest number of coding genes based on NCBI annotation.The aim is to extract the maximum core genes. In order to achieve this goal, we choose \textit{Zea Mays NC\_001666.2} to be our reference genome.
+\item Build Blast database for the rest of unannotated genomes.
+\item Compare reference Genes: based on the genomes in the database. We querying each reference gene with the database by using \textbf{Blastn}. The result with alignment scores for each gene will store in separated file.
+\item Generate match table: In this table, each row represent referenced genes, while columns represent genomes. To fill this table, a developed code will open each output file for reference genes and extract the number of genomes and a list of genomes names where gene sequence have hits.
+\end{enumerate}
-\subsubsection{Core Genes from Dogma Annotation}
+The core genome can be extracted from the table by taking as possible the maximum number of genes that exists in the maximum number of genomes.
\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 or genes sequences 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 quiality 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 previous methods.
+
+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 Predict quality genomes: the process is to pick a genome annotation from two techniques, extracting all common genes based on genes names, then applying Needle-man wunch algorithm to align the two sequences based on a specific threshold. 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 them and vise-versa. Using first method to extract core genes will be sufficient because we already check their sequences.
+\item Display tree: An evolution tree then will be display based on the intersections of quality genomes.
+\end{enumerate}
\ No newline at end of file
