본 연구에서는 가리맛조개 (S. constricta)의 토사물을 현미경 검경과 차세대염기서열분석 (NGS) 기법으로 먹이원을 확인하고, 이를 통해 형태학적 및 분자학적 방법에 따른 먹이원 분석을 비교하였다. 가리맛조개 (S. constricta)의 먹이원은 분석방법에 따라 차이를 보였다. 먹이원생물은 위 내에서 분해되어 현미경 분석을 통한 생활사 확인과 정량적 분석이 가능하였으나 형태학적 및 해부학적 특성 파악이 불완전하였다. NGS 분석은 유기물 형태로 잔존하는 생물의 DNA 확인이 가능하여 현미경 검경 결과와의 상호보완적 적용 가능성을 확인하였다.

Recent increase of raw sequences generated by next generation sequencing (NGS) machines enabled re-analyzing raw sequences for diverse purposes: one is assembling organelle genome. One recent study completed the mitochondrial genomes of 14 ants from public raw sequences in subfamily Pseudomyrmecinae not having organelle genomes. Along with this approach, we have found four ant species of which genome papers were published and its raw sequences were open to public but its mitochondrial genome has not been assembled yet: Harpegnathos saltator and three Pogonomyrmex species (P. rugosus, P. anergismus, and P. colei). We assembled four complete mitochondrial genomes, presenting mitogenome of H. saltator 16,467 bp long and those of three Pogonomyrmex species above 21 kb long, ranking top among all known Hymenopteran mitogenomes. Four mitochondrial genomes contain 13 PCGs, 22 tRNAs, and 2 rRNAs, conserved as in all other insects. Phylogenomic tree based on partial or complete mitogneomes covering 26 genera provides insights of ant mitogenomic phylogeny and evolution.

산화아연나노물질은 상업적으로 가장 폭넓게 사용되고 있는 물질 중 하나이다. 특히 식품산업에서 산화아연나노물 질은 아연에서 기인하는 세포생장과 면역증진 등의 효과를 나타낼 뿐 아니라 산화아연자체에 의한항균 기능성을 보유 하고 있어 건강보조식품, 식품첨가물 및 식품포장재 등에 다양하게 이용되고 있다. 이와 같이 식품산업에서 산화아연 나노물질의 사용이 증가함에 따라 나노물질에 대한 노출이 증가하고 있는데, 상대적으로 나노물질의 안전성에 대한 연구는 미흡한 편이다. 최근 들어 산화아연나노물질의 독성시험이 세포와 동물모델에서 이루어지고 있으나 나노물질 의 독성과 입자크기 및 체내 성상에 대한 상관관계는 명확히 규명되지 않은 실정이다. 따라서 본 연구에서는 산화아 연나노물질(nano ZnO)을 랫드에 14일 반복 경구 투여 후 차세대 염기서열분석기법(next generation sequencing, NGS)을 이용하여 간 조직에서의 전사체 발현양상을 분석하였으며, 입자크기와 이온화도에 따른 독성을 비교하기 위하여 벌크 크기의 산화아연(bulk ZnO)과 염화아연(ZnCl2)을 동일한 방법으로 비교 평가하였다. 그 결과 산화아연의 입자크기 및 이온화도에 따라 서로 다른 유전자 발현양상을 보였다. 특히 아연 이온 투여에 의한 전사체 발현 양상이 입자에 의한 양상과는 상이한 것으로 나타나 입자크기 및 체내 성상에 따라 잠재적 독성영향이 달라질 수 있음을 제시하였다. 본 연구결과는 산화아연나노물질의 잠재적 경구 독성의 평가와 독성메커니즘의 예측 자료로써 유용하게 활용될 수 있을 것으로 기대된다.

In recent years, high-throughput next-generation sequencing (NGS) techniques have provided fascinating opportunities to understand the biology of non-model organisms, especially insect species. The decrease in sequencing costs and extensive sequencing services from NGS providers has brought many entomologists to be involved in genome sequencing. However, poor planning can lead to extremely fragmented genome assemblies which prevents high quality gene annotation and other desired analyses. Insect genomes can be problematic to assemble, due to combinations of high polymorphism, inability to breed for genome homozygosity, and small physical sizes limiting the quantity of DNA able to be isolated from a single individual. Given to the rapid development of host resistance to multiple classes of insecticides, it is indispensable to study the comprehensive genomic information of insects. Recent advances in sequencing technology and assembly strategies can able to fetch breakthroughs in deciphering the genetic information of insects. Here, we present the cost effective high throughput genome sequencing and assembly strategies for insect species in respects to taxonomy, evolutionary history, immune response, drug development, insect host-virus interactions and pest management etc.

White-backed planthopper, Sogatella furcifera (Horvath) (Hemiptera: Delphacidae), has been a serious migratory pest in Korea. It is important to figure out the migration route and gene flow of S. furcifera. Microsatellite marker (SSR) shows high efficiency as molecular markers. Unfortunately, various microsatellite marker of S. furcifera has not been developed to see genetic diversity. S. furcifera samples were collected from Vietnam, Laos and three different sites of Bangladesh in 2012. We extracted DNA by using QIAamp DNA Mini Kit and ran next generation DNA sequencer (NGS) Roche 454 to develop a new microsatellite marker. Roughly, about 18 singleton primers and 14 contigs primers were found. We will test these primers with S. fucifera DNA samples, and figure out the accurate new microstatellite marker.

NGS(Next Generation Sequencing) 기법이 2007년부터 적용된 이후 약 10년도 되지 않은 짧은 기간 동안 유전체 연구에 상당히 큰 변화가 일어나고 있다. 유전자 중심의 연구에서 유전체를 기반으로 하는 형태로 연구 패러다임이 변화되고 있으며, 컴퓨터 하드웨어의 급속한 성능 증가와 더불어 생물정보의 중요성이 다시 부각되고 있다. 인간 중심의 유전체 연구는 식물과 동물, 미생물로 급격하게 분야가 확장되고 있으며, 한 대의 시퀀싱 머신에서 120Gb의 시퀀싱 데이터가 단 29시간만에 만들어지는 기술적 성과를 확인할 수 있다. 곤충 분야에서는 i5K(The 5,000 Insect Genome Project) 국제 컨소시엄이 시작되었으며, BGI(Beijing Genome Institute)에서도 다양한 곤충의 유전체 시퀀싱 및 분석 사업을 진행하겠다고 제안하고 있다. 국내에서도 차세대바이오그린21사업의 동물유전체육종사업단에서 가축유전체와 더불어 몇 가지의 곤충 유전체 연구가 진행되고 있으며, 금년부터 시작되는 농촌진흥청의 다부처유전체사업에서도 일반작물, 원예작물, 약용작물, 가축과 더불어 곤충분야도 한 부분을 차지하게 되었다. 아직은 다른 분야에 비해서 잘 알려져 있지 않지만, 곤충 분야 연구의 잠재성을 검토해 볼 때 가장 빠르게 유전체 연구를 통해 유용한 결과를 획득할 수 있으리라 생각된다. 하지만, 곤충 분야의 유전체 연구를 위해서는 선행되었던 다른 유전체 연구의 문제점을 파악하여 접근하여야 효율적인 성과를 도출할 수 있을 것이다. 따라서, 곤충 유전체 연구에 대한 동향과 생물정보학적인 기법 등을 제안하고자 한다.

The application to genome study has been particularly developed with the introduction of the next-generation DNA sequencer (NGS) Roche/454 and Illumina/Solexa systems, along with bioinformation analysis technologies of whole-genome de novoassembly, expression profiling, DNA variation discovery, and genotyping. One of the advantages of the NGS systems is the cost-effectiveness to obtain the result of high-throughput DNA sequencing for genome, RNAnome, and miRNAnome studies. Both massive whole-genome shotgun paired-end sequencing and mate paired-end sequencing data are important steps for constructing de novo assembly of novel genome sequencing data and for resequencing the samples with a reference genome DNA sequence. To construct high-quality contig consensus sequences, each DNA fragment read length is important to obtain de novo assembly with long reading sequences of the Roche/454 system. It is necessary to have DNA sequence information from a multiplatform NGS with at least 2× and 30×depth sequence of genome coverage using Roche/454 and Illumina/Solexa, respectively, for effective an way of de novo assembly, as hybrid assembly for novel genome sequencing would be cost-effective. In some cases, Illumina/Solexa data are used to construct scaffolds through de novo assembly with high coverage depth and large diverse fragment mate paired-end information,even though they are already participating in assembly and have made many contigs. Massive short-length reading data from the Illumina/Solexa system is enough to discover DNA variation, resulting in reducing the cost of DNA sequencing. MAQ and CLC software are useful to both single nucleotide polymorphism discovery and genotyping through a comparison of resequencing data to a reference genome. Whole-genome expression profile data are useful to approach genome system biology with quantification of expressed RNAs from a whole-genome transcriptome, depending on the tissue samples, such as control and exposed tissue. The hybrid mRNA sequences from Rohce/454 and Illumina/Solexa are more powerful to find novel genes through de novo assembly in any whole-genome sequenced species. The 20× and 50× coverage of the estimated transcriptome sequences using Roche/454 and Illumina/Solexa, respectively,is effective to create novel expressed reference sequences. However, only an average 30× coverage of a transcriptome with short read sequences of Illumina/Solexa is enough to check expression quantification, compared to the reference expressed sequence tag sequence. In an in silicomethod, conserved miRNA and novel miRNA discovery is available on massive miRNAnome data in any species. Particularly, the discovered target genes of miRNA could be robust to approach genome biology study.

Recent years have seen the introduction of next-generation sequencing (NGS) technologies and their use in the fields of bioscience and biotechnology. Not only has the availability of NGS technologies revolutionized the way genome research is carried out, the massive sequence data produced by NGS technologies have also been driving the advancement in bioinformatics required for downstream analysis, storage, and accessibility. However, the processing of NGS data is still challenging owing to such properties as the shortness of reads, the sheer amount of data, and the low base qualities. For high-quality de novo genome sequencing, we used a hybrid approach that utilizes Sanger end sequencing of fosmid libraries for the scaffolding of NGS-derived contigs. This strategy was successfully applied to the genome sequencing of microbial cell factories requiring a complete gene list for the metabolic pathway, and a regulatory network for the design and development of industrial strains. Recently, software developments have facilitated de novo genome assembly which can produce genome scaffolds from short reads only. Automatic gap closing, which incorporates paired-end short reads into preexisting scaffolds, is also feasible. High-throughput multiplexed genome sequencing based on the Illumina platform has become a routine task for genome prospecting and the comparative genomic analysis of useful microbial strains. We also carried out the resequencing of E. coli strains generated from a combined approach of a long-term experimental evolution and proton beam-induced mutagenesis. NGS-based resequencing allows the identification of genetic variations from multiple samples at a lower cost, and the tracing of evolutionary pathways that are engraved in the genomic sequences. NGS-based transcriptome sequencing (RNA-seq), which is becoming popular as a substitute for traditional microarray experiments, also provides evidence for the identification of genes from novel genomes. Recent achievements in genome sequencing and analysis of eukaryotic microbes will also be introduced.

Background : The advancement of next-generation sequencing technology dramatically reduces the cost for sequencing and it contributes to create a new research environment that utilizes large amount of genome sequences to answer many biological questions. With this new research trend, reference genome sequences of several major crops have been released to the research community and utilized in various researches in agriculture. Coupled with molecular breeding technology, NGS based genome research will possibly allow selecting a new plant material possessing useful traits in early stage and efficiently developing a superior cultivar. Methods and Results : The objectives of this research are to collecting various genetic variations (SNPs, indels and TE mediated variations) in major and minor crops, to develop molecular markers using NGS based genomic data (resequencing, GBS, transcriptome), and to develop a visualization tools to enhance the utility of the NGS data. Currently major analysis pipelines have been developed to detect SNPs, indel and polymophic SSRs using whole genome and transcriptome data, and a pipeline for identification of MITE insertion polymorphism is under development. In addition to that, for orphan crop, we also implemented an efficient and robust method to assemble a complete chloroplast, mitochondria and 45S rDNA using low coverage whole genome data in order to develop an inter- and intra-specific molecular barcode markers. Conclusion : NGS provide a new level of researches in many crop plants. Large amount of genomic information provides an opportunity to understand domestication and genetic variations, and to develop a better crop for future.

The rice recombinant inbred lines derived from Milyang23 and Gihobyeo cross were used in genetic mapping and QTL analysis studies. In this study, we developed a new 101 CAPS markers based on the SNPs in the whole genome region between these varieties. As a result, the total genetic distance and average distances were 1,696.97 cM and 3.64 cM, respectively. In comparison to the distance of the previous genetic map constructed based on 365 DNA markers, the new genetic map was found to have a decreased distance. The map was applied for the detection of QTLs on all seven traits relevant to diameter of stem internode, length of culms, length of panicles and the number of panicles including the correlation analysis between each trait. The QTLs results were similar to the report in previous studies, whereas the distance between the markers was narrowed and accuracy increased with the addition of 101 CAPS markers. A total of 9 new QTLs were detected for stem internode traits. Among them, qI1D-6 had higher LOD of 5.1 and phenotype variation of 50.92%. In this experiment, a molecular map was constructed with CAPS markers using next generation sequencing showing high accuracy for markers and QTLs. In the future, developing more accurate QTL information on stem internode diameters with various agriculturally important traits will be possible for further rice breeding.

Blueberry (Vaccinium spp.) is a member of the Ericaceae and eleven varieties have been registered at the Korea Seed & Variety Service for Plant Variety Protection (PVP). This study was to develop simple sequence repeat (SSR) markers next generation sequencing (NGS) analysis and to analysis genetic relationship of blueberry 31 varieties. Highbush blueberry ‘Camellia’ and rabbiteye blueberry ‘Alapaha’ varieties were used as sequencing materials. Out of total 987 SSR primers detected between ‘Camellia’ and ‘Alapaha’, 148 SSR primers were initially applied to select SSR markers for identification of blueberry varieties. Fourteen SSR markers showed polymorphism between 8 varieties. Seven SSR markers showed reproducibility and clear peak among 14 SSR markers. Genetic relationships of 31 blueberry varieties were analyzed and identified using 7 SSR markers. A total of 30 polymorphic SSR alleles were obtained and two to seven alleles were detected for each locus with an average of 4.3 alleles per locus. Average polymorphism information content was 0.556, ranging from 0.374 to 0.714. Genetic distance of clusters ranged from 0.38 to 0.93 by unweighted pair-group method with arithmetical average based on Jaccard’s distance coefficients. These newly developed SSR markers indicate usefulness for variety identification related to seed dispute and distinctness, uniformity and stability (DUS) test for blueberry.

The Asteraceae/Compositae family is one of the biggest families in flowering plants and has more than 23000 species including the economically important lettuce, sunflower, and chicory as well as the agronomic weeds. With its significance and the progress in sequencing technology, its species have been subjected to the genome sequencing project worldwide. Although chrysanthemum is an important plant in the floricultural industry, however, it has been less studied at the level of genomics, compared with other species in the Asteraceae. There were only several reports on comparative analysis of transcriptome for chrysanthemum. Actually, the genome of Chrysanthemum species is known to be gigantic and complex with diverse status ranging from diploid to decaploid. Since the cultivated and commercial chrysanthemum exhibits hexaploid genome, we decided to select the diploid species with smaller genome as a material for reference genome sequencing. Thus, we launched a genome sequencing project with C. boreale which was previously reported to be diploid by cytogenetic analysis. We constructed sequencing libraries with insert size 300bp and 500bp and sequenced them from the paired end in 100bp read length with Illumina’s HiSeq platform. After quality checking, we preprocessed raw reads by removing duplicated reads and trimming reads with low quality value. Kmer frequency analysis with the cleaned reads showed that the genome is heterozygous, highly repetitive and gigantic, ranging from 2.9Gb to 5.8Gb. The cleaned reads were further subjected to error correction and primary assembly with SOAPdenovo2. Here, we’ll report the result of Kmer frequency analysis and genome assembly.

Anthocyanin is known for positive health beneficial effects that including reduces age related oxidative stress and inflammatory responses. It was produced by vegetable crops and a lettuce is one of the crops. The general pathway of anthocyanin expression is well defined but it is not clear how environments effects on anthocyanin accumulation in a lettuce. Therefore we initiated to study interaction between anthocyanin expression and environment factors. Frist, we applied RGB leaf images in a lettuce to calculate anthocyanin areas in a leaflet with two different cultivars, different developmental stages, and different environments. Later, we attempted to capture RNA expression level with next generation sequence (NGS) RNA sequencing method called RNA-seq. As a result, combined two technologies showed that quantitate phenotypic data help to understand the gene expression of anthocyanin in lettuce cultivars.

The NGS technologies of genome DNA structure, expression profiling and epigenome elements have been used widely as approaches in the expertise of genome biology and genetics. The application to genome study has been particularly developed with the introduction of the next-generation DNA sequencer (NGS) Roche/454, Illumina/Solexa and PacBio systems along with bioinformation analysis technologies of whole-genome de novo assembly, expression profiling, DNA variation discovery, and genotyping. One of the advantages of the NGS systems is the cost-effectiveness to obtain the result of high-throughput DNA sequencing for genome, RNAnome, and miRNAnome studies. Both massive whole-genome shotgun paired-end sequencing and mate paired-end sequencing data are important steps for constructing de novo assembly of novel genome sequencing data and for resequencing the samples with a reference genome DNA sequence. To construct high-quality contig consensus sequences, each DNA fragment read length is important to obtain de novo assembly with long reading sequences of the Roche/454 and PacBio systems. It is necessary to have DNA sequence information from a multiplatform NGS with at least 2x and 30x depth sequence of genome coverage using Roche/454 and Illumina/Solexa, respectively, for effective an way of de novo assembly, as hybrid assembly for novel genome sequencing would be cost-effective. In some cases, Illumina/Solexa data are used to construct scaffolds through de novo assembly with high coverage depth and large diverse fragment mate paired-end information, even though they are already participating in assembly and have made many contigs. Massive short-length reading data from the Illumina/Solexa system is enough to discover DNA variation, resulting in reducing the cost of DNA sequencing. MAQ and CLC software are useful to both SNP discovery and genotyping through a comparison of resequencing data to a reference genome. Whole-genome expression profile data are useful to approach genome system biology with quantification of expressed RNAs from a whole-genome transcriptome, depending on the tissue samples, such as control and exposed tissue. The long read sequence data of PacBio are more powerful to find full length cDNA sequence through de novo assembly in any whole-genome sequenced species. An average 30x coverage of a transcriptome with short read sequences of Illumina/Solexa is enough to check expression quantification, compared to the reference EST sequence. In an in silico method, conserved miRNA and novel miRNA discovery is available on massive miRNAnome data in any species. Particularly, the discovered target genes of miRNA could be robust to approach genome biology study.