• Plant Breeding and Biotechnology
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• v.5 no.4
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• pp.269-281
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• 2017

With the continual development of genetically modified (GM) crops, it has become necessary to develop detailed and effective molecular characterization methods to select candidate events from a large pool of transformation events. Relative to traditional molecular analysis methods such as the polymerase chain reaction (PCR) and Southern blot hybridization, next generation sequencing (NGS) technology for whole-genome sequencing of complex crop genomes had proven comparatively useful for in-depth molecular characterization. In this study, four transformation events, including one in Bacillus thuringiensis (Bt)-resistant rice, one in resveratrol-producing rice, and two in beta-carotene-enhanced soybeans, were selected for molecular characterization. To merge NGS analysis and Southern blot-hybridization results, we confirmed the transgene insertion sites, insertion construction, and insertion numbers of these four transformation events. In addition, the read-coverage depth assessed by NGS analysis for inserted genes might provide consistent results in terms of inserted T-DNA numbers in case of complex insertion structures and highly duplicated donor genomes; however, PCR-based methods can produce incorrect conclusions. Our combined method provides an effective and complete analytical approach for whole-genome visual inspection of transformation events that require biosafety assessment.

• Genomics & Informatics
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• v.13 no.3
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• pp.81-85
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• 2015

Molecular characterization technology in genetically modified organisms, in addition to how transgenic biotechnologies are developed now require full transparency to assess the risk to living modified and non-modified organisms. Next generation sequencing (NGS) methodology is suggested as an effective means in genome characterization and detection of transgenic insertion locations. In the present study, we applied NGS to insert transgenic loci, specifically the epidermal growth factor (EGF) in genetically modified rice cells. A total of 29.3 Gb (${\sim}72{\times}coverage$) was sequenced with a $2{\times}150bp$ paired end method by Illumina HiSeq2500, which was consecutively mapped to the rice genome and T-vector sequence. The compatible pairs of reads were successfully mapped to 10 loci on the rice chromosome and vector sequences were validated to the insertion location by polymerase chain reaction (PCR) amplification. The EGF transgenic site was confirmed only on chromosome 4 by PCR. Results of this study demonstrated the success of NGS data to characterize the rice genome. Bioinformatics analyses must be developed in association with NGS data to identify highly accurate transgenic sites.

• Animal Systematics, Evolution and Diversity
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• v.36 no.4
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• pp.336-346
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• 2020

Mitochondrial genome is an important molecule for systematic and evolutionary studies in metazoans. The development of next-generation sequencing (NGS) technique has rapidly increased the number of mitogenome sequences. The process of generating mitochondrial genome based on NGS includes different steps, from DNA preparation, sequencing, assembly, and annotation. Despite the effort to improve sequencing, assembly, and annotation methods of mitogenome, the low quality and/or quantity sequence in the final map can still be generated through the work. Therefore, it is necessary to check and curate mitochondrial genome sequence after annotation for proofreading and feedback. In this study, we introduce the pipeline for sequencing and curation for mitogenome based on NGS. For this purpose, two mitogenome sequences of Dermatobranchus otome were sequenced by Illumina Miseq system with different amount of raw read data. Generated reads were targeted for assembly and annotation with commonly used programs. As abnormal repeat regions present in the mitogenomes after annotation, primers covering these regions were designed and conventional PCR followed by Sanger sequencing were performed to curate the mitogenome sequences. The obtained sequences were used to replace the abnormal region. Following the replacement, each mitochondrial genome was compared with the other as well as the sequences of close species available on the Genbank for confirmation. After curation, two mitogenomes of D. otome showed a typically circular molecule with 14,559 bp in size and contained 13 protein-coding genes, 22 tRNA genes, two rRNA genes. The phylogenetic tree revealed a close relationship between D. otome and Tritonia diomea. The finding of this study indicated the importance of caution and curation for the generation of mitogenome from NGS.

• Journal of Genetic Medicine
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• v.12 no.2
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• pp.109-117
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• 2015

Purpose: Mitochondrial diseases are clinically and genetically heterogeneous disorders, which make their exact diagnosis and classification difficult. The purpose of this study was to identify pathogenic mitochondrial DNA (mtDNA) mutations in 2 Korean families with myoclonic epilepsy with ragged-red fibers (MERRF) and Leigh syndrome, respectively. Materials and Methods: Whole mtDNAs were sequenced by the method of mtDNA-targeted next-generation sequencing (NGS). Results: Two causative mtDNA mutations were identified from the NGS data. An m.8344A>G mutation in the tRNA-Lys gene (MT-TK) was detected in a MERRF patient (family ID: MT132), and an m.9176T>C (p.Leu217Pro) mutation in the mitochondrial ATP6 gene (MT-ATP6) was detected in a Leigh syndrome patient (family ID: MT130). Both mutations, which have been reported several times before in affected individuals, were not found in the control samples. Conclusion: This study suggests that mtDNA-targeted NGS will be helpful for the molecular diagnosis of genetically heterogeneous mitochondrial diseases with complex phenotypes.

• Microbiology and Biotechnology Letters
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• v.49 no.3
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• pp.440-448
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• 2021

Helicobacter pylori (H. pylori) establishes long-term infections associated with severe gastric diseases such as peptic ulceration and gastric cancer. Exposure to an antibacterial agent can help regulate the expression levels of its pathogenic genes. In this study, we analyzed the transcriptional changes in H. pylori genes induced by β-caryophyllene. We used next-generation sequencing (NGS) to analyze RNA expression changes, and reverse transcription-polymerase chain reaction (RT-PCR) was performed as required to verify the results. The NGS results showed that 30 out of 1,632 genes were expressed differentially by β-caryophyllene treatment. Eleven genes associated with DNA replication, virulence factors, and T4SS components were significantly downregulated. RT-PCR confirmed that treatment reduced the expression levels of 11 genes. RT-PCR showed the reduced expression of 11 genes (dnaE, dnaN, holB, gyrA, cagA, vacA, secA, flgE, virB2, virB4, and virB8) following β-caryophyllene treatment. These results suggest that β-caryophyllene can modulate various H. pylori pathogenic determinants and be a potential therapeutic agent for H. pylori infection.

• Genomics & Informatics
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• v.14 no.3
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• pp.78-84
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• 2016

Extranodal natural killer (NK)/T-cell lymphoma, nasal type (NKTCL), is a malignant disorder of cytotoxic lymphocytes of NK or T cells. It is an aggressive neoplasm with a very poor prognosis. Although extranodal NKTCL reportedly has a strong association with Epstein-Barr virus, the molecular pathogenesis of NKTCL has been unexplored. The recent technological advancements in next-generation sequencing (NGS) have made DNA sequencing cost- and time-effective, with more reliable results. Using the Ion Proton Comprehensive Cancer Panel, we sequenced 409 cancer-related genes to identify somatic mutations in five NKTCL tissue samples. The sequencing analysis detected 25 mutations in 21 genes. Among them, KMT2D, a histone modification-related gene, was the most frequently mutated gene (four of the five cases). This result was consistent with recent NGS studies that have suggested KMT2D as a novel driver gene in NKTCL. Mutations were also found in ARID1A, a chromatin remodeling gene, and TP53, which also recurred in recent NGS studies. We also found mutations in 18 novel candidate genes, with molecular functions that were potentially implicated in cancer development. We suggest that these genes may result in multiple oncogenic events and may be used as potential bio-markers of NKTCL in the future.

• Parasites, Hosts and Diseases
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• v.54 no.6
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• pp.751-758
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• 2016

This study aimed at constructing a draft genome of the adult female worm Toxocara canis using next-generation sequencing (NGS) and de novo assembly, as well as to find new genes after annotation using functional genomics tools. Using an NGS machine, we produced DNA read data of T. canis. The de novo assembly of the read data was performed using SOAPdenovo. RNA read data were assembled using Trinity. Structural annotation, homology search, functional annotation, classification of protein domains, and KEGG pathway analysis were carried out. Besides them, recently developed tools such as MAKER, PASA, Evidence Modeler, and Blast2GO were used. The scaffold DNA was obtained, the N50 was 108,950 bp, and the overall length was 341,776,187 bp. The N50 of the transcriptome was 940 bp, and its length was 53,046,952 bp. The GC content of the entire genome was 39.3%. The total number of genes was 20,178, and the total number of protein sequences was 22,358. Of the 22,358 protein sequences, 4,992 were newly observed in T. canis. Following proteins previously unknown were found: E3 ubiquitin-protein ligase cbl-b and antigen T-cell receptor, zeta chain for T-cell and B-cell regulation; endoprotease bli-4 for cuticle metabolism; mucin 12Ea and polymorphic mucin variant C6/1/40r2.1 for mucin production; tropomodulin-family protein and ryanodine receptor calcium release channels for muscle movement. We were able to find new hypothetical polypeptides sequences unique to T. canis, and the findings of this study are capable of serving as a basis for extending our biological understanding of T. canis.

• Microbiology and Biotechnology Letters
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• v.50 no.2
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• pp.301-309
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• 2022

Helicobacter pylori (H. pylori) establishes infection in the human gastric mucosa for a long time and causes severe gastric diseases such as peptic ulcer and gastric cancer. When H. pylori is exposed to the antibacterial agents or inhibitors, the expression of pathogenic associated genes could be altered. In this study, we analyzed the transcriptional changes of H. pylori genes induced by zerumbone treatment. RNA expression changes were analyzed using next-generation sequencing (NGS), and then reverse transcription-polymerase chain reaction (RT-PCR) was performed to verify the results. As a result of NGS analysis, a total of 23 out of 1,632 genes were differentially expressed by zerumbone treatment. RT-PCR confirmed that zerumbone treatment regulated the expression level of 14 genes. Among the genes associated with DNA replication, transcription, virulence factors and T4SS components, 10 genes (dnaE, dnaQ, rpoA, rpoD, secA, flgE, flhA, virB5, virB8 and virB9) were significantly down-regulated and 4 genes (flaA, flaB, virB4 and virD4) were up-regulated. The results of our current study imply that zerumbone might be a potential therapeutic agent for H. pylori infection by regulating factors related to various H. pylori pathogenicity.

• 한국균학회소식:학술대회논문집
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• 2015.05a
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• pp.53-53
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• 2015

Agrobacterium tumefaciens-mediated transformation(ATMT) of Flammulina velutipes was used to produce a diverse number of transformants to discover the functions of gene that is vital for its variation color, spore pattern and cellulolytic activity. Futhermore, the transformant pool will be used as a good genetic resource for studying gene functions. Agrobacterium-mediated transformation was conducted in order to generate intentional mutants of F. velutipes strain KACC42777. Then Agrobacterium tumefaciens AGL-1 harboring pBGgHg was transformed into F. velutipes. This method is use to determine the functional gene of F. velutipes. Inverse PCR was used to insert T-DNA into the tagged chromosomal DNA segments and conducting sequence analysis of the F. velutipes. But this experiment had trouble in diverse morphological mutants because of dikaryotic nature of mushroom. It needed to make monokaryotic fruiting varients which introduced genes of compatible mating types. In this study, next generation sequencing data was generated from 28 strains of Flammulina velutipes with different phenotypes using Illumina Hiseq platform. Filtered short reads were initially aligned to the reference genome (KACC42780) to construct a SNP matrix. And then we built a phylogenetic tree based on the validated SNPs. The inferred tree represented that white- and brown- fruitbody forming strains were generally separated although three brown strains, 4103, 4028, and 4195, were grouped with white ones. This topological relationship was consistently reappeared even when we used randomly selected SNPs. Group I containing 4062, 4148, and 4195 strains and group II containing 4188, 4190, and 4194 strains formed early-divergent lineages with robust nodal supports, suggesting that they are independent groups from the members in main clades. To elucidate the distinction between white-fruitbody forming strains isolated from Korea and Japan, phylogenetic analysis was performed using their SNP data with group I members as outgroup. However, no significant genetic variation was noticed in this study. A total of 28 strains of Flammulina velutipes were analyzed to identify the genomic regions responsible for producing white-fruiting body. NGS data was yielded by using Illumina Hiseq platform. Short reads were filtered by quality score and read length were mapped on the reference genome (KACC42780). Between the white- and brown fruitbody forming strains. There is a high possibility that SNPs can be detected among the white strains as homozygous because white phenotype is recessive in F. velutipes. Thus, we constructed SNP matrix within 8 white strains. SNPs discovered between mono3 and mono19, the parental monokaryotic strains of 4210 strain (white), were excluded from the candidate. If the genotypes of SNPs detected between white and brown strains were identical with those in mono3 and mono19 strains, they were included in candidate as a priority. As a result, if more than 5 candidates SNPs were localized in single gene, we regarded as they are possibly related to the white color. In F. velutipes genome, chr01, chr04, chr07,chr11 regions were identified to be associated with white fruitbody forming. White and Brown Fruitbody strains can be used as an identification marker for F. veluipes. We can develop some molecular markers to identify colored strains and discriminate national white varieties against Japanese ones.