Whole Microbial Genome Assembly And Analysis Based On Ion Torrent Sequencing Data

Microorganism is very tiny(diameter less than 0.1 mm) and its structure is simple but closed related with our life, including bacteria, actinomycetes, protozoa, algae and protozoa. As a carrier of genetic information, microbial genome is the core code for people deciphering the biological characters of microorganism. The study of microbial genome is very significant for people understanding microbial genetic information,microbial metabolic processes and some special features such as drug resistance and pathogenicity. Obtaining the complete genome sequence should be a priority for all in the study of microbial genome.With the rapid development of high-throughput sequencing technology, the time and costs of DNA sequencing continue to decrease and produce higher data volumes than ever before. Whole microbial genome sequencing is also developing at a speed unheard-of before. However, the short length of high-throughput sequencing read makes it difficult for the whole genome sequence assembly. There are three current mainstream HTS platform: Roche’s 454 GS FLX +, Illumina’s Hiseq and Miseq, Life’s Ion Torrent.Among of them, Ion Torrent is the fastest sequencing platform that have a simple operation and lower cost,which is the best choice for medium-scale sequencing and very suitable for whole microbial genome sequencing.In this paper, based on Ion Torrent sequencing data, we have made a comprehensiveexploration of the sequence assembly of whole microbial genome and the corresponding solutions for encountered problems in the exploration. Whole genome sequence assembly is similar to building blocks or playing Rubik’s cube. It is a process of reconstructing a complete genome sequence using the sequenced DNA fragments.Based on the characteristics of Ion Torrent sequencing data, we firstly made a selection of assembly algorithms and software,。Finally,we chose Newbler as the assembly software which launched by Roche 454 l with OLC algorithm. Then during the subsequent process of completing map of microbial genome, according to the characteristics of the microbial genome assembly, we have selected three genome assembly assistive tools. Amone of them, Contig Scape used to display the complex relationship of contigs generated by microbial genome assembly program, so that we could be familiar with the basic information about the genomic structure quickly;optical mapping of genome used to guide sequence assembly, it had a significant increasing for the quality of microbial genome sequence assembly, and it could also correct the assembly result; Gap Filler genome used to filling internal gap of genome.Comprehensive the above approach, we have established a method for microbial genome sequence assembly based on Ion Torrent sequencing data. we also write some personalized program designed for simplifying the process, which is helpful for those researchers who have no genome assembly experience.In addition, for the problems that repetitive DNA sequences confuse genome sequence assembly, here we show that a new method with high price-performance ratio to resolve the issue, which is use of the real-time polymerase chain reaction(PCR). Through designing a simple primers, PCR amplification and observing the CT values, we could distinguish the relationship of contig directly. It took less time and energy.More importantly of than all of that, it had a lower cost, which had significance for genome assembly, especially microbial genome sequence assembly. At the end of thispaper, we illustrated briefly the personalized analysis of microbial genomes with three examples: COG annotation, comparison of gene content of different strains, identifying virulence genes.In summary, we further improved the process of microbial genome sequence assembly on the basis of the existing methods using Ion Torren sequencing data. Besides, a new way was found to resolve the problem caused by repeats during assembly in this paper,and we also introduced some subsequent analysis of the whole microbial genome. Each method mentioned in this paper is applied to an examples. However, for different subjects and requirements, researchers still need to analyze case-by-case. I hope paper work will give helpful references to other researchers.