Analysis Of Several Viral And Bacterial Genomics By Ion Torrent Sequencing Platform And Bioinformatics

Virus, a kind of special life, is strictly parasitic on live cells including human,animal, plant, insect, fungus and bacteria etc.75%of human infectious diseases werecaused by virus.A replication circle of virus includes: entering (or inject its DNA into) the host byrecognizing the receptor on the surface of the host cell, replicating its genomic DNA,synthesizing the offspring protein, packaging, lysing from the host cell and infectingnew host cell for another circle.Bacteriophage is the virus of bacteria, which has almost similar replication circlewith other viruses. For a dsDNA phage, it cyclizes its genome with the help of theterminal repeats or the cohensive ends of its genome, then it takes the rolling circlereplication mode to replicate and produce a lot of concatemeric DNA, which serve asthe substrate of the small subunit of the terminase to perform the recognizing andcleavage process to produce daughter DNA. The daughter DNA is packaged into theprohead by the large subunit of the terminase, and then ultimately become the genomeof the next generation virion. The replication packaging process is vital to the virus inthe replication circle. Hence, research of virus will focus on these two processes:replication and pachaging. Meanwhile, these two processes are closely related to theterminal sequence of the vrus genome: replication needs cyclizing its genome with thehelp of the repeats at the genome termini and packaging can produce new termini toassist the next replication. Hence, it is important to characterize the terminal sequenceof a virus genome. However, traditional method were time consuming and easy tolose some important detail of the genome termini. Therefore, a high throughput,accurate, rapid termini detection method was required.Iridovirus is a dsDNA virus, which belongs to the family Iridoviridae. The virusparticle of iridovirus display purple and blue rainbow when exposed to the obliquelight, thus it is called the irido. Iridovirus and Chloriridovirus in Iridoviridae weredefined as Invertebrate iridoviruses (IIVs), which mainly infect the insects. Importantinfluences of IIVs against insects, like sub-lethal effects and inducing apoptosis, etc.Hence, the IIVs could be potentially utilized for pest control. Unfortunately, thefunctional activities associated with the virus and interactions between virus and hostcells remain to be elucidated. Furthermore, there were only4complete genomes ofIIVs in the GenBank, therefore, the whole genome sequencing of IIVs is required.The complete sequence can provide guidance for the genetic and other kind offunction research of Iridoviridae. Bacillus anthracis is an etiological agent for worldwide zoonotic diseases and isone of the most dangerous bioterrorism agents. The1993unsuccessful anthrax aerosolattack in Japan and the2001letter attacks in the United States making it verychallenging to rapidly and accurately discriminate naturally occurring outbreaksversus intentional release of micro-organisms. Efficient genotyping methods aredemanded not only for biosecurity related issues including helping microbial forensicsto trace back the origin of an aggression and investigating the worldwide bacterialdistribution, but also for facilitating the epidemiological studies to reduce the risk ofan outbreak or control it effectively.B. anthracis exhibits very high interspecies genome homogeneity and very lowgenetic diversity that strongly hinders the efforts to discriminate among strains forphylogenetic and forensic purposes. Even with20-40generations’infection-death-infection cycle, the genetic composition of the B.anthracis genomestill remained largely unchanged.Two molecular genome typing systems, SNP (single nucleotide polymorphism)and MLVA (multiple locus variable number tandem repeat analysis) had greatlyfacilitated the discrimination of B.anthracis strains with relatively high resolution.However, SNP has bias and the number of SNPs between very close strains was veryfew. MLVA results fluctuated remarkably in different labs. Therefore, typing andsource tracing method with higher resolution and more stability was required forissues such as infectious diseases and terrorist incidents caused by B. anthracis.Morganella morganii (Mm) is a Gram-negative anaerobic bacteria and the onlyspecies in the genus Morganella, which belongs to the family Enterobacteriaceae. Mmpresents natural drug resistance to antibiotics β-lactamases. It is a kind ofopportunistic pathogenic enterobacterium which can cause severe infection, and it cancaused complex infections of the urinary in nosocomial settings. It has been causallylinked to catheter-associated bacteriuria, postoperation wound infection of nosocomialinfection. It can cause inflammation like diarrhea, septicemia, pneumonia, myositis,cephalitis, pericarditis, chorioamnionitis, entophthalmia and idiopathic bacterialperitonitis. The morbidity of the infection by Mm was increased year by year. Mm hasbecome one of the most common pathogenic bacteria of the acquired infection in thehospital.High-throughput sequencing (HTS) has the characteristic of high throughput,accuracy, speed and low cost. It is widely used in various fields of biomedicalresearch, including re-sequencing the genome of species with reference genome, DeNovo sequencing the genome of species without reference genome, detection of novelviruses by sequencing of small RNA, and transcriptome studies.At present, the main HTS platforms include Miseq and Hiseq2000of Illumina,454genome sequencer of Roche, and Ion Torrent PGM of Life technologies. The platforms of Illumina have the characteristics of accuracy and high throughput, andthey become the most popular deep sequencing machines, although it has shorterreads and longer time. Roche454has longer reads but is very expensive. Ion Torrentis both quick and cheap, and can serve as a practical tool for bacterial and viralgenome sequencing, as well as for the functional and the characteristical research formicrobes.Our findings based on next generation sequencing of bacteriophage and viralgenomes revealed that HFSs are indeed the viral genome termini. Further, we usedNGS data i) to determine simultaneously the complete sequence of viral genomes aswell as their terminal sequences, ii) to establish a criterion to distinguish the type oftermini and the viral packaging mode that exist in each bacteriophage and virus, iii) todetect additional details of terminal sequences such as terminal repeats, secondarytermini and multi-termini, iv) to assess the ends of the genome of iridovirus strainAMIV, which has highly preferred sequences instead of random sequences as inT4-like phages, v) N4-like phages with short and variable length direct terminalrepeats with a unique sequence at the left genome termini and two sequences at theright genome termini. Using these techniques, we determined for the first time thepresence of a unique left end and random right ends in S. aureus phages IME-SA1,IME-SA2, and IME-SA3, and the same terminase cleavage site and terminal structurein S. aureus phages. In addition, we also proposed a formula to calculate the terminalrepeat length. The method we propose thus simplifying the termini analysis.Theoretically, this application can be further extended to analysis of genomes of plantand animal viruses. This study proposed a novel and efficient method for research onviral replication, packaging, terminase activity, transcription regulation, andmetabolism of the host cell.The Iridovirus of AMIV was isolated from the mosquitoes collected fromYunnan province in2012. The homogenate was added to the C3/36cell culture andCPE was checked. The CPE can be observed after three blind passages. Then weperform massive culture, concentration, purification and genome extraction for AMIV.After subject AMIV to shotgun and matepair sequencing, i）we obtained the wholegenome sequence, ii）after the BLAST analysis, we confirm the virus was aniridovirus with very new nucleotide sequence, iii）we discovered that AMIV has verylow homology with other iridovirus by comparative genome analysis, iv） thephylogenetic analysis results show AMIV was a novel genotype, v）we found AMIVvirions have a diameter of about180nm with an unclear icosahedral contour. Eachparticle was surrounded by a thin layer of intermediate lipid membrane, vi）wecharacterized the detailed CPE process by fluorescence microscope observation, and the C6/36cells showed a significant CPE starting from about9hours after infectionwith AMIV and almost all the cells were lysed within36hours, suggesting apathogenic infection of C6/36by AMIV, vii）The genome of AMIV has terminalredundancy (TR) with about10%of the genome length, this ensures that each virionreceives at least one copy of every gene, the phenomenon may have an evolutionaryadvantage, viii）The reason for the rapid replication of AMIV was analyzed, and wefound there are12copies of bro-like gene which is highly conserved and associatedwith the replication and transcription of host DNA. It is reported that ascovirus canreplicate very rapid since it has three11copy of the bro-like gene. In addition, only54genes with known function were identified in AMIV genome, and interestly,20(about40%) of them were associated with replication, also suggesting the replicationof virus DNA be efficient.In August2012, there was an outbreak of anthrax in Liaoning Province, northeastof China, in which7people were infected and many cows were killed. One B.anthracis isolate named Han was isolated from one dead cow, and it exhibited minorpathogenicity on mice. It is speculated that this strain Han might be resulted from thereverse mutation of a locally used attenuated anthrax vaccine strain Vac. In order totell if the isolate Han and the vaccine strain Vac are actually the same, both of thestrains were subjected to high throughput sequencing. With the genome sequencingdata, canonical SNP analysis and whole genome SNP screening were performed. Theresults indicate that strain Han was remarkably different from the Vac strain. Furtheranalysis by MLVA showed that Han clustered with previously reported Chinesestrains. In addition, a sequence-based MLVA as a mend of traditional MLVA wasdeveloped to distinguish the very closely related strains and to get highersource-tracking resolution. The sequence-based MLVA also confirmed that strain Hanis a naturally occurred isolate instead of a terrorist attack agent deliberately distributed.In conclusion, this study, with complete genome sequencing technique and ultra-highresolution source-tracking sequence-based MLVA, demonstrated that the anthraxoutbreak was caused by a local B. anthracis strain. The applied strategy not onlyfacilitates the epidemiological studies, but also enhances the discrimination ofnaturally occurring outbreaks versus intentional release of pathogens.At the end of this study, shotgun and mate-pair sequencing method was adoptedto sequence the whole genome of Mm which is isolated along with B. anthracis strainHan. We obtained the complete genome of Mm by De Novo assembly and thiscomplete genome sequence represent the second complete sequence of Mm, and thefirst complete genome sequence of Mm isolated in Mainland China. In addition, weannotated genome, and eventually discovered432pathogenicity-related genes, whichmay help future studies on the pathogenic mechanism of this bacteria.