• Journal of Ginseng Research
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• v.33 no.1
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• pp.55-58
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• 2009

Generally, the direct sequencing through PCR is faster, easier, cheaper, and more practical than clone sequencing. Frequently, standard PCR amplification is usually interpreted by mispriming internal or external regions of the target template. Normally, DNA fragments were eluted from the gel using Gel extraction kit and subjected to direct sequencing or cloning sequencing. Cloning sequencing has often troublesome and needs more time to analyze for many samples. Since touchdown (TD) PCR can generate sufficient and highly specific amplification, it reduces unwanted amplicon generation. Accordingly, TD PCR is a good method for direct sequencing due to amplifying wanted fragment. In plants the 5S-rRNA gene is separated by simple spacers. The 5S-rRNA gene sequence is very well-conserved between plant species while the spacer is species-specific. Therefore, the sequence has been used for phylogenetic studies and species identification. But frequent occurrences of spurious bands caused by complex genomes are encountered in the product spectrum of standard PCR amplification. In conclusion, the TD PCR method can be applied easily to amplify main 5S-rRNA and direct sequencing of panax ginseng cultivars.

• Animal Bioscience
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• v.36 no.2_spc
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• pp.364-373
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• 2023

The gastrointestinal (GI) tract of ruminants contains diverse microbes that ferment various feeds ingested by animals to produce various fermentation products, such as volatile fatty acids. Fermentation products can affect animal performance, health, and well-being. Within the GI microbes, the ruminal microbes are highly diverse, greatly contribute to fermentation, and are the most important in ruminant nutrition. Although traditional cultivation methods provided knowledge of the metabolism of GI microbes, most of the GI microbes could not be cultured on standard culture media. By contrast, amplicon sequencing of 16S rRNA genes can be used to detect unculturable microbes. Using this approach, ruminant nutritionists and microbiologists have conducted a plethora of nutritional studies, many including dietary interventions, to improve fermentation efficiency and nutrient utilization, which has greatly expanded knowledge of the GI microbiota. This review addresses the GI content sampling method, 16S rRNA gene amplicon sequencing, and bioinformatics analysis and then discusses recent studies on the various factors, such as diet, breed, gender, animal performance, and heat stress, that influence the GI microbiota and thereby ruminant nutrition.

• Journal of Animal Environmental Science
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• v.21 no.3
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• pp.93-98
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• 2015

The objective of this study was to review application of next-generation sequencing (NGS) to investigate microbiome in the livestock sector. Since the 16S rRNA gene is used as a phylogenetic marker, unculturable members of microbiome in nature or managed environments have been investigated using the NGS technique based on 16S rRNA genes. However, few NGS studies have been conducted to investigate microbiome in the livestock sector. The 16S rRNA gene sequences obtained from NGS are classified to microbial taxa against the 16S rRNA gene reference database such as RDP, Greengenes and Silva databases. The sequences also are clustered into species-level OTUs at 97% sequence similarity. Microbiome similarity among treatment groups is visualized using principal coordinates analysis, while microbiome shared among treatment groups is visualized using a venn diagram. The use of the NGS technique will contribute to elucidating roles of microbiome in the livestock sector.

• Environmental Engineering Research
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• v.14 no.1
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• pp.3-9
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• 2009

Microorganisms play an important role in the geochemical cycles, industry, environmental cleanup, and biotechnology among other fields. Given the high microbial diversity, identification of the microorganism is essential in understanding and managing the processes. One of the most popular and powerful method for microbial identification is comparative 16S rRNA gene analysis. Due to the highly conserved nature of this essential gene, sequencing and later comparison of it against known rRNA databases can provide assignment of the bacteria into the taxonomy, and the identity of its closest relatives. Isolation and sequencing of 16S rRNA genes directly from natural environments (either from DNA or RNA) can also be used to study the structure of the whole microbial community. Nowadays, novel sequencing technologies with massive outputs are giving researchers worldwide the chance to study the microbial world with a depth that was previously too expensive to achieve. In this article we describe commonly used research approaches for the study of individual microorganisms and microbial communities using the tools provided by Ribosomal Database Project website.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.4
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• pp.1739-1743
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• 2014

MicroRNAs are a class of small noncoding RNA which play important regulatory roles in a variety of cancers. MiRNA-specific expression profiles have been reported for several pathological conditions. In this study, we combined large scale parallel Solexa sequencing to identify 11 up-regulated miRNAs and 19 down-regulated miRNAs with computational techniques in the sera of ovarian cancer patients while using healthy serum as the control. Among the above, four miRNAs (miR-22, miR-93, miR-106b, miR-451) were validated by quantitative RT-PCR and found to be significantly aberrantly expressed in the serum of ovarian cancer patients (P<0.05). There were no significant differences between samples from cancer stage I/II and III/IV. However, the levels of miR-106b (p=0.003) and miR-451 (p=0.007) were significantly different in those patients under and over 51 yearsof age. MiR-451 and miR-93 were also specific when analyzed with reference to different levels of CA125. This study shows that Solexa sequencing provides a promising method for cancer-related miRNA profiling, and selectively expressed miRNAs could be used as potential serum-based biomarkers for ovarian cancer diagnosis.

• Journal of Aquaculture
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• v.10 no.4
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• pp.403-407
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• 1997

Intraspecific sequence variation was detected by polymerase chain reaction (PCR) and direct sequencing of a 350-nucleotide region of the mitochondrial 12S rRNA gene of two natural populations (Han River and Nakdong River) and one hatchery stock (Jinhae Inland Fisheries Institute) of local strain common carp, one Israeli strain of common carp stock from Pukyong National University (PKU), and one hybrid between Israeli strain of common carp female and local strain common carp male from PKU stock. There is little variation in 350 bases of the mitochondrial 12S rRNA gene sequences among 2 natural and 1 hatchery local strain common carp populatins, representing abut 7 to 20 nucleotide differences (less than 6%). The sequence of specimens from Han River was more similar to that from Nakdong River (identity=98.0%) than to that from Jinhae Inland Fisheries Institute (identity=96.3%). Sequence variation between Israeli strain and wild local strain common carp was higher than the variation within natural stocks. The level of variation was ranged from 15.7 to 17.7%. The hybrid showed very similar nucleotide4 sequence of 12S rRNA gene to the sequence of Israeli strain with the identity of 98.9%.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.33 no.2
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• pp.103-109
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• 2000

Complete senuences of the mitochondrial rRNA Benes were determined among six salmonines in Korean Waters (Brachpmystax lenok, Onoorhpchus keta, O. masou mason, O. mason ishikawae, O. mykiss, and albino mutant of O. mykiss). The purposes of this study were to provide the basic information on levels of mtDNA polymorphism among these species for genetic characterization; discuss phylogentic relationships among three Oncorhynchus sepecies; demonstrate the utility of rRNA gene sequence data as a genetic marker for disringuishinf among Korean salmonines. PCR/direct sequencing data indicated the following consistent results; 1) 12S rRNA genes was 945 bases long in Oncorhynchus species, and 946 bases in B. lenot including one insertion. 2) Of sequence variation in mitochondrial rRNA regions, transitional substitutions were superior to transversion. 3) The significant differences were not shown in the intraspecific variation values in these gene regions. The percentage sequence divergence values were ranged from $0.066 to 0.212{\%}$. 4) The interspecific divergences were greater than the intraspecific variation. Nevertheless, ribosomal RMh genes were more conserved among species than the other mitochondrial genes, and they showed potentiality as an intergenic marker for systematics. In addition, phylogenetic trees, constructed from this data, supported that cherry salmon was closer to chum salmon than to rainbow trout, and that lenok was most distantly related species in six salmonid species.

• Genomics & Informatics
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• v.18 no.3
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• pp.26.1-26.6
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• 2020

Single-cell RNA sequencing (scRNA-seq) has been widely applied to provide insights into the cell-by-cell expression difference in a given bulk sample. Accordingly, numerous analysis methods have been developed. As it involves simultaneous analyses of many cell and genes, efficiency of the methods is crucial. The conventional cell type annotation method is laborious and subjective. Here we propose a semi-automatic method that calculates a normalized score for each cell type based on user-supplied cell type-specific marker gene list. The method was applied to a publicly available scRNA-seq data of mouse cardiac non-myocyte cell pool. Annotating the 35 t-stochastic neighbor embedding clusters into 12 cell types was straightforward, and its accuracy was evaluated by constructing co-expression network for each cell type. Gene Ontology analysis was congruent with the annotated cell type and the corollary regulatory network analysis showed upstream transcription factors that have well supported literature evidences. The source code is available as an R script upon request.

• Korean Journal of Microbiology
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• v.35 no.2
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• pp.121-127
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• 1999

An effective computer program for assembling fragments in DNA sequencing has been developed. The program, called SeqEditor (Sequence Editor), is usable on the pcrsonal computer systems of MS-Widows which is the mosl popular operating system in Korea. It c'm recd several sequence file formats such as GenBak, FASTA, and ASCII. In the SeqEditor program, a dynamic programming algorihm is applied to compute the maximalscoring overlapping alignment between each pjlr of fragments. A novel feature of the program is that SeqEdilor implemnents interaclive operation with a graphical user interface. The performance lests of the prograln 011 fragmen1 data from 16s and 18s rDNA sequencing pi-ojects produced saiisIactory results. This program may be useful to a person who has work of time with large-scale DNA sequencing projects.

• Journal of Microbiology and Biotechnology
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• v.28 no.7
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• pp.1112-1121
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• 2018

Jeotgal is a Korean traditional fermented seafood with a high concentration of salt. In this study, we isolated lactic acid bacteria (LAB) from galchi (Trichiurus lepturus, hairtail) and myeolchi (Engraulis japonicas, anchovy) jeotgal on MRS agar and MRS agar containing 5% NaCl (MRS agar+5% NaCl), and identified them by using 16S rRNA gene sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as culture-dependent methods. We also performed polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) as a culture-independent method to identify bacterial communities. Five samples of galchi-jeotgal and seven samples of myeolchi-jeotgal were collected from different regions in Korea. A total of 327 and 395 colonies were isolated from the galchi- and myeolchi-jeotgal samples, respectively. 16S rRNA gene sequencing and MALDI-TOF MS revealed that the genus Pediococcus was predominant on MRS agar, and Tetragenococcus halophilus on MRS agar+5% NaCl. PCR-DGGE revealed that T. halophilus, Tetragenococcus muriaticus, and Lactobacillus sakei were predominant in both types of jeotgal. T. halophilus was detected in all samples. Even though the same species were identified by both culture-dependent and -independent methods, many species identified by the culture-dependent methods were not in the bacterial list identified by the culture-independent methods. The distribution of bacteria in galchi-jeotgal was more diverse than in myeolchi-jeotgal. The diverse LAB in galchi- and myeolchi-jeotgals can be further studied as candidates for starter cultures to produce fermented foods.