The advent of Next Generation Sequencing (NGS) technology has changed the research paradigm and become an essential tool for recent biological and medical study. In today’s market, there are several sequencing platforms which have specific sequencing principle, and the result of each sequencing platform has different characteristics among them. Hence, each sequencing method became more specialized for specific research purpose, and researchers who consider NGS analysis have to understand the very basic characteristics of each NGS platform. NGS is used in various studies and they are usually classified into 5 categories (Re-sequencing, RNA-seq, de novo assembly, Metagenomics and Epigenomics) of analysis. In this session, we will introduce the characteristics of sequencing platforms and examples of recent research on each of the 5 analysis categories. In addition, we will talk about the benefit of NGS study compared to the traditional study and how these NGS technologies can be applied in developmental biology research.

Common buckwheat (F. esculentum) and tartaryan buckwheat, also called as bitter buckwheat (F. tartarycum) grain and leaves (buds) are used in various dietary preparations and as leafy vegetable. The cultivated area of buckwheat is increased based on its nutritional value. Particularly bitter buckwheat is a rich source of rutin compared to common buckwheat which helps in reducing intra-vascular cholesterol, high blood pressure, diabetes and is also reported to have a crucial role in pharmaceutical research. With this functional characteristics of bitter buckwheat, the cultivation is now highly increased. But a few genetic and genomic research of tartari buckwheat are published until now. Here we described the complete full chloroplast genome sequence with NGS. Tartary buckwheat complete chloroplast genome is composed of a total sequence length of 159,272 bp which is 327 bp lesser than common buckwheat genome of 159,599 bp. Large single copy region (LSC) is comprised of 84,398 bp in tartary and 84,888 bp in common buckwheat whereas small single copy region (SSC) is 13,292 bp and 13,343 bp and the size of inverted repeat region (IR) is 61,532 bp and 61,368 bp in tartary and common buckwheat respectively. Total RNA bases were 11942 and 11950 and overall GC-content in tartary and common buckwheat is almost similar which is 37.9% and 38% with a GC skew of -0.016 and 0.02 respectively. Total repeat bases accounted for 1,056 bp and 804 bp with an average repeat length of 48 bp and 45 bp and the length of an average intergenic distance was 495 bp and 502 bp in tartary and common buckwheat respectively. F. tarataticum cp genome has a total of 104 genes including 82 protein coding genes, 29 transfer RNA genes and 4 ribosomal RNA genes. Protein coding genes include photosynthesis related genes majorly in addition to transcription and translation related genes. LSC region has 62 protein coding genes and 22 tRNA genes whereas SSC region contains 11 protein coding genes and one tRNA gene. The nucleotide and amino acid sequences of protein coding genes in LSC, SSC and inverted repeat regions in F.tartaricum and F.esculentum are highly similar with a total average identity of 98.8 and 98.3% respectively.

Generally, the virus was detected by the ELISA using the serological method and RT-PCR based on the genetic information. Recently, NGS (next-generation nucleotide sequencing) has been used in genome analysis and diseases diagnostics. To identify distribution aspects of viruses, we collected diseased samples twice in soybean breeding field. After extraction of total RNA from the collected bulk samples, RNA was sequenced by the NGS method. The NGS data were analyzed using the bioinformatics software. With newly produced NGS data, the identification of distribution aspects of organisms in field was estimated in this study. Sequence based identification method should be more accurate diagnostic tools of the target diseases and be able to predict occurrence of potential and new pathogens. NGS method will also provide the basic data by identifying the distribution of using bacteria. In this study, we analyzed the extracted RNA from the collection of approximately 3000 samples. Consequently, we confirmed the following types: the 7,089 kinds of bacteria including Burkholderiaceae, the 13,397 kinds of Eukaryota, the 952 kinds of viruses from the first bulk samples, the 4,160 kinds of bacteria including Burkholderiaceae, the 10,475 kinds of Eukaryota, and the 576 kinds of viruses from the second bulk samples

최근 등장한 차세대 유전체 해독 기술(NGS, Next-generation sequencing)은 대용량의 데이터를 짧은 시간 내에 생산하는 것을 가능케 한다. 이 기술을 이용하여 whole genome de novo assembly, re-sequencing, transcriptome, epigenome 등 다양한 응용 분야로 발전하고 있으며, 이 정보를 해석하여 단일 염기 서열 변이 분석과 유전자 발현량 분석, 후성 유전체 연구 등의 유전체 수준에서 연구를 가능하게 하고 있다. 특히 NGS 중 전사체 해독 기술(RNA-seq)은 유전자의 발현량에 대한 정밀한 분석과 선택적 스플라이싱에 의한 새로운 전사체 발견, 신규 유전자 발견 등 기존의 방법들이 제공하지 못하는 새로운 정보를 밝혀내고 있으며, 후성 유전체 해독 연구 역시 생물의 시기별 환경별 유전자 발현 조절 기작인 메틸레이션에 대한 다양한 분석을 가능하게 하여 다양한 환경에서 발현되는 유전자의 isoform과 그 발현량 조절에 대한 연구를 가능하게 한다. 이 발표에서는 NGS 기술에 대한 설명과 이를 이용한 다양한 응용분야를 소개하고, NGS 기술을 이용하여 이루어진 발생생물학 분야에서의 최근 연구 결과에 대한 소개한다.

고추 탄저병은 국내에서 아주 피해가 심한 병 중의 하나로 본 연구팀은 십수 년 동안 탄저병 저항성에 대해 유전분석을 수행하는 동시에 저항성 품종 육성에 노력을 기울여 왔다. 이전에 사용하였던 탄저병 저항성 소재는 Capsicum baccatum 종의 PBC81 accession이었는데, 이와 가장 교잡화합성이 높았던 C. annuum 종의 SP21 계통을 모친으로 사용하여 종간 교잡을 수행하였고, 이에 대한 BC1F1과 BC1F2 분리집단에서 QTL mapping을 수행하여 두 가지의 탄저병(Colletotrichum acutatum과 C. capsici)에 대한 각각의 저항성 주동 QTL을 탐색함과 동시에 연관된 분자표지를 개발하였다. 본 연구에서는 탄저병 저항성 소재로 PBC81이 아닌 PI594137과 AR을 사용하여 NGS re-sequencing을 수행한 후 대량의 SNP를 탐색하고자 하였다. PI594137은 C. baccatum 종에 속하며, PBC81보다 좀 더 broad spectrum resistance를 보인다. AR은 AVRDC에서 분양 받은 재료인데, C. chinense Jacq. PBC932의 열성 저항성을 C. annuum에 도입한 계통이다. 탄저병 저항성 QTL mapping은 Golden aji(C. baccatum, 탄저병 이병성)와 PI594137의 F2 분리집단과 SP211(C. annuum, 탄저병 이병성)과 AR의 F2 분리집단에서 수행할 계획이어서 각각의 양친 사이(Golden aji vs. PI594137과 SP211 vs. AR)에서 SNP를 탐색하였다. NGS re-sequencing을 통해 읽혀진 염기서열 총 길이는 PI594137이 40.5Gbp, Golden aji가 12.1Gbp, AR이 12.8Gbp, SP211이 11.5Gbp였다. 이 염기서열을 사용하여 생물정보학적 분석((주)씨더스에 의뢰)을 수행하였는데, PI594137과 Golden aji 사이에서 333,816개, AR과 SP211 사이에서 1,218,595개의 SNP를 최종적으로 탐색할 수 있었다. 탐색된 SNP는 탄저병 저항성 QTL mapping 분석에 유용하게 사용될 수 있을 것이다.

As soybean (Glycine max) is known for its high nutritional value of oil and protein, soybean has been domesticated and cultivated by one specific character trait based on human selection. Importantly, tracing back in time where G. max and G. soja, the undomesticated ancestor of G. max have diverged plays an important role in studying of genetic diversity and in investigating the common ancestor of soybean. In this study, we sequenced 6 G. max and 6 G. soja using Illumina’s Hiseq 2000 with a low coverage sequencing technology to estimate the divergence of times between genotypes and populations. A total of the 12 genotypes were sequenced at the average depth of 6.5 and resulted 892.5 Mb and 903.3 MB consensus sequences with the coverage of 91.54% and 92.65% for G. max and G. soja, respectively. The whole genome SNP analysis showed that G. max had lower frequency levels of polymorphism (~0.1%) than G. soja (~0.25%). And, a high number of SNPs located in introns were found among 6 G. soja genotypes as SNPs were approximately twice than those found in 6 G max. The number of SNPs in G. max intronic regions was 53,134, whereas a total of 133,329 SNPs were discovered in G. soja introns. Almost an equal number of SNPs were discovered in 5’ UTR and exon regions; however, different numbers of SNP in CDS and 3′ UTR were identified. By the rate of nonsynonymous change, divergence of time between G. soja and G. max would be investigated.

Genetic map provides basic and important informations for breeding. Therefore, genetic map construction is a essential process in plant research. Panax ginseng is one of the most famous medical plant in the world. However, genetic informations of this medical plant for breeding are not enough. Because of little informations, genetic map construction of panax ginseng provides very useful information for breeding. Using Solexa next generation sequencing (NGS) technology, we have been produced a lot of expressed sequence tags (ESTs) and whole genome sequences from Chunpoong (368 Gb) and Yunpoong (6 Gb) cultivar. To develop large amount of DNA markers and thus construct high resolution genetic map, we inspect large scale of SSR motif and putative SNP sites which can be used as dCAPs markers using produced ginseng’s sequence data. As a result, we can find a number of DNA markers that have polymorphism between Yunpoong and Chunpoong cultivar. These developed DNA markers were analyzed for F2 population of Yunpoong x Chunpoong to find markers showing Mendelian segregation ratio 1:2:1.