understanding the evolutionary relationships between groups of organisms has become increasingly reliant on phylogenetic analysis .
comparative sequence analysis is a useful method by which one can identify gene , infer the function of a gene 's product , and identify novel functional elements . by comparing several sequences along their entire length , researchers can find conserved residues that are likely preserved by natural selection .
a reading frame is a set of consecutive , nonoverlapping triplets of three consecutive nucleotides .
a codon is a triplet equating to an amino acid or stop signal during translation .
an open reading frame ( orf ) is the section of reading frame containing no stop codons .
a protein can not be made if rna transcription ceases prior to reaching the stop codon .
therefore , to ensure that the stop codon is translated at the correct position , the transcription termination pause site is located after the orf .
orfs also have been utilized to classify various virus families [ 13 ] , including members of norovirus [ 3 , 4 ] .
the open reading frame finder ( orf finder )   is a graphical analysis tool that searches for open reading frames in dna sequences .
the orf investigator   program provides information on the coding and noncoding sequences and performs pairwise alignment of different dna regions .
this tool efficiently identifies orfs and converts them to amino acid codes , declaring their respective positions in the sequence .
first , the dna sequence is transcribed into rna , and all potential orfs are identified .
these orfs are encoded within each of the six translation frames ( 3 in the forward direction and 3 in the reverse direction ) , so that users can identify the translation frame yielding the longest protein coding sequence .   
several biological organizations have implemented bioinformatics tools on websites . the national center for biotechnology information ( ncbi )  
provides many tools for comparing database - stored nucleotide or protein sequences , including the well - known blast algorithms .
ncbi also provides several databases , such as genbank and snp , in which biologists can seek homology or specific functions .
the european molecular biology laboratory ( embl )   provides freely available data and online bioinformatics tools to all facets of the scientific community .
cloud computing is a recently developed concept that delivers computing resources , either hardware or software , over the internet .
many types of cloud computing have been proposed , such as infrastructure as a service ( iaas ) , platform as a service ( paas ) , software as a service ( saas ) , network as a service ( naas ) , and storage as a service ( staas ) .
most of these services rely on virtualization technology  the creation of virtual hardware platforms , operating systems , storage devices , and network resources .
cloud computing is welcomed for its user friendliness , virtualization , internet - centric focus , resource variety , automatic adaptation , scalability , resource optimization , pay - per - use , service slas ( service - level agreements ) , and infrastructure slas .
many cloud computing vendors distribute these resources on demand from large resource pools installed in data centers .
amazon ec2   supplies an infrastructure service , while google app engine   and microsoft 's azure services platform   supply platform services . in academia ,
hadoop   is an open - source software framework that supports data - intensive distributed computation . under hadoop
the computational task is divided into many small tasks , each of which may be executed or reexecuted on a compute node in the hadoop cluster .
mapreduce also provides a distributed file system , the hadoop distributed file system ( hdfs ) , that stores the data on compute nodes , enabling a very high aggregate bandwidth across the cluster . both map / reduce and the distributed file system are robust against failure
several sequence analysis tools have been redeveloped as cloud tools based on the hadoop architecture , such as cloudblast   and crossbow .
such importing of preexisting tools constitutes the main goal of bioinformatics as a service ( baas ) . in this paper
, we develop a high - availability , large - scale orf phylogenetic analysis cloud service based on virtualization technology and hadoop .
this service provides phylogenetic analyses from orfs based on hadoop clusters to support multiple requests .
the physical computing power is regarded as a user - pays utility that users can request as desired .
users can upload their sequence data or files through the master node ( web portal ) and then submit a job . the job is assigned to the slave node containing the uploaded data , and the slave node completes the job .
since orf comparisons have unambiguously established the homology of norovirus , we here adopt norovirus as a case study .
the results show that the proposed cloud - based analysis tool , by virtue of virtualization technology and hadoop framework , can readily facilitate baas .
in this paper , we propose a cloud - based orf phylogenetic analysis service combining hadoop framework , virtualization technology , phylogenetic tree tool , and diversity analysis .
hadoop is performed on the vms created by virtualization technology such as kernel - based virtual machine ( kvm ) .
the underlying architecture ensures elasticity , scalability , and availability of the proposed cloud - based service .  
the proposed cloud service integrates the orf finding process , phylogenetic tree contractions , and orf diversity analysis to generate a complete phylogenetic analysis .
the procedure of the analysis is outlined below and shown in figure 1 . 
 step  1 : detecting open reading frames .
the orf finder locates all open reading frames of a specified minimum size in a sequence . in this study ,
this tool identifies all open reading frames using the standard or alternative genetic codes .  

step  2 : constructing phylogenetic tree based on open reading frames . a phylogenetic tree ( or evolutionary tree )
is a branching ( tree ) diagram showing the inferred evolutionary relationships between biological species or other entities based on similarities and differences in their physical and/or genetic characteristics .
virus orf alignments might reveal a common viral ancestor or an orf that is common to all viruses .
this algorithm builds two phylogenetic trees ; one based on full sequences and the other for orfs only , thereby revealing the variance between the two trees .  

small diversity value at a position implies that protein sequences are very similar at that position .
such high - variance frames can be used to observe protein structural differences and to aid vaccine development . in this paper ,
diversity is calculated from the entropy as follows : 
 ( 1)h(i)=p(xi)log2p(xi ) ,  xi  = { g , a , i , v ,  } , 
 where h(i ) is the value of entropy and p(xi ) is the probability of finding a specified amino acid at position i. to find the significant position , entropy values under a certain threshold are filtered out . in this study ,
the cloud platform for proposed phylogenetic analysis tool is constructed on two important technologies : virtualization and the hadoop framework .
the scalability and availability are guaranteed by hdfs , a self - healing distributed storage system and mapreduce , a specific fault - tolerant distributed processing algorithm .
the master node consists of a job tracker , task tracker , name node , and data node .
a slave node , or computing node , comprises a data node and a task tracker .
the job tracker assigns map / reduce tasks to specific nodes within the cluster , ideally those already containing the data or at least within the same rack .
a task - tracker node accepts map , reduce , and shuffle operations from a job - tracker .
hadoop also creates multiple replicas of data blocks and distributes them to data nodes throughout a cluster to enable reliable , extremely rapid computations .
the name node serves as both a directory namespace manager and a node metadata manager for the hdfs .
the hdfs allows the data to spread across hundreds or thousands of nodes or machines , and the tasks are computed on data - holding nodes .
hadoop replicates data , so that if one replica is lost , backup copies exist .
when a node fails during computation , hadoop restarts the halted task on another node containing replicate data . in the hadoop framework ,
node failures are detected using the heartbeat mechanism , by which individual task nodes ( task trackers ) constantly communicate with the job tracker .
if a task tracker fails to communicate with the job tracker for a period of time , the job tracker will assume that the task tracker has crashed .
the job tracker knows which task trackers ( data nodes ) contain replicate data , and it issues a restart task . in this paper , the proposed cloud service was implemented by combining hadoop cluster distribution with a management model . in our cloud server , a submitted job is computed in a data node . rather than processing parallel data ,
therefore , submitted data are distributed to a data node by the hdfs , while the computing process is delivered to the task tracker and copied with the submitted data .
the usual goal of virtualization is to improve scalability and overall hardware - resource utilization .
virtualization permits the parallel running of several operating systems on a single physical computer . while a physical computer in the classical sense constitutes a complete and actual machine , a virtual machine ( vm ) is a completely isolated machine running a guest operating system within the physical computer . to ensure scalability and efficiency , all components  job tracker , task tracker , name node , and data node  in our cloud service operate as virtual machines .  
cloud - based orf phylogenetic analysis service was developed on a virtualization platform with the hadoop framework as described above .
the master node ( name node ) and slave node ( data node ) are the master vm and slave vm , respectively .
when a phylogenetic analysis request is submitted , it is saved in a job queue .
the master node periodically extracts the jobs from the job queue and assigns them to slave nodes ( or mappers ) , which perform the task . at the completion of all jobs
, the reducer collects the results and saves them in the network file system storage ( nfs ) .
a single comparison result of a phylogenetic job is saved in a single file of nfs .
as shown in figure 5 , a data node running in vm2 performs a phylogenetic analysis and a name node runs in vm1 .
the reducer , running in vmn+2 , collates the results from the data nodes executing the phylogenetic analyses . in this service , the user uploads protein sequences and submits a phylogenetic analysis request on the website portal .
all submitted analysis jobs are gathered in the job queue and sequence data are stored in different hosts by hdfs .
the analysis results are sent to both data node and reducer to produce the final result stored in nfs .
users either enter the comparative dna / rna sequences on the web portal or upload a file containing comparative rna sequences from a web portal .  
 step  2 : sequence translation . to detect the orf regions , all input rna sequences are translated to protein sequences based on the genetic code .
the genetic code is the set of rules by which rna sequence information is translated into proteins .
each codon in an rna sequence usually represents a single amino acid specified by the corresponding genetic code .
this step identifies the functional orfs , recall that significant orfs are rare . in our service , the user can provide the length of orf that he / she regards as meaningful .
, the first orf ( denoted as ab447445_1 ) extends from positions 3 to 5099 in the sequence ab447445 . in this step ,
two types of phylogenetic trees are built , one using the full sequence length and the other using orfs only . from the three orf regions identified in the analysis , three orf phylogenetic trees are built .
these trees are recorded in ph format and are then transferred to and stored in the portal . meanwhile ,
step  4 : report result . in this step , the ph formatted trees are drawn as three diagrams and displayed on the portal .
each server was equipped with two quad - core intel xeon 2.26  ghz cpus , 24  gb ram , and 296  gb hard disk , running under the ubuntu operating system version 10.4 , with 8 virtual machines on each server .
one vm constituted the job tracker and name node ; the others are task trackers and data nodes .
two of these vms are name node and data node running the reducer ; the remaining six are responsible for map operation . for the experiment
, we randomly produced three datasets , each containing 20 sequences of different lengths ( 300 , 400 , and 600 nucleotides ) .
clustalw and the proposed service were applied three times , for simulating three orf phylogenetic analyses .  
the computation time of the proposed service illustrated in figure 8 is proportional to the number of mappers .
the execution time is considerably reduced when six mappers are used , relative to two mappers .  
figure 9 compares the performance between sequential phylogenetic analysis methods such as clustalw and the proposed service with six mappers , for different sequence lengths .
clearly , the proposed service in the hadoop framework achieves better performance than standard sequential phylogenetic analysis .
the nov genome is a single - stranded , positive sense , polyadenylated rna encoding three open reading frames , orf1 , orf2 , and orf3 .
orf1 encodes a long polypeptide that is cleaved intracellularly into six proteins by the viral proteinase .
orf2 encodes a viral capsid protein , vp1 , while orf3 encodes a vp2 protein that is regarded as a minor structural component of virus particles , apparently responsible for the expression and stabilization of vp1 . like the majority of rna viruses ,
the virus is tentatively divided into five genogroups and more than 25 genotypes , based on similarities between orf2 sequences [ 33 , 34 ] .
therefore , the homology of this type of virus may be identified from orf similarities . identifying this homology will assist in viral drug and vaccine design .
the phylogenetic trees constructed from full length sequences and three orfs are shown in figure 10 . obviously , these trees differ from each other .
the tree constructed from the full length sequences ( figure 10(a ) ) demonstrates an evolutionary relationship between the viruses
. however , different orfs yield distinctly different trees ( figures 10(b)10(d ) ) , suggesting that viruses can copy orfs from other viruses and alter their function by integrating them into their own sequences .
therefore , by establishing evolutionary relationships for each orf , virologists can analyze the diseases caused by specific orfs .  
the residue position of high entropy is provided in figure 12 , which shows four phylogenetic trees and the diversity bar graph .
the positions ( also the amino acids ) of high diversity are shown in the box .
cloud computing is the online delivering of computing resources , such as hardware and software .
users can access cloud - based applications through a web browser or via applications on mobile devices .
although many bioinformatics tools have been developed as web applications , these are typically deployed in a server , which has limited computing power .
currently , some tools have been redeveloped as distributed computing tools based on the hadoop framework .
these tools are readily deployed on a cluster provided by a cloud computing vendor such as amazon ec2 .
deployment of preexisting tools to the cloud environment is the current trend of bioinformatics as a service .   in this paper
, we propose a high - scale , available cloud - based open reading frame phylogenetic analysis service based on a hadoop cluster using virtualization technology .
virtualization enables the proposed service to copy large quantities of jobs . because hadoop is strongly buffered against faults , the proposed cloud service guarantees that submitted jobs are recovered by task reassignment , ensuring a high - availability cloud service .
our case study demonstrated that our service can construct different phylogenetic trees from comparisons of different orfs .
 phylogenetic analysis has become essential in researching the evolutionary relationships between viruses . 
 these relationships are depicted on phylogenetic trees , in which viruses are grouped based on sequence similarity . 
 viral evolutionary relationships are identified from open reading frames rather than from complete sequences . 
 recently , cloud computing has become popular for developing internet - based bioinformatics tools . 
 biocloud is an efficient , scalable , and robust bioinformatics computing service . in this paper , we propose a cloud - based open reading frame phylogenetic analysis service . 
 the proposed service integrates the hadoop framework , virtualization technology , and phylogenetic analysis methods to provide a high - availability , large - scale bioservice . in a case study 
 , we analyze the phylogenetic relationships among norovirus . 
 evolutionary relationships are elucidated by aligning different open reading frame sequences . 
 the proposed platform correctly identifies the evolutionary relationships between members of norovirus . 
1. Introduction
2. Methods
3. Experiment
4. Case Study
5. Conclusion