• 대한의생명과학회지
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• 제22권4호
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• pp.150-159
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• 2016

Knowledge of the distribution and biodiversity of environmental bacteria and the ecosystem that influences them is crucial for predicting an ecosystem. However, bacterial culture methods can only analyze approximately 0.1% of the existing microorganisms, those that are readily cultured under laboratory conditions. By contrast, next-generation sequencing (NGS) has generally been known to obtain more diverse profiling of bacterial composition. We compared the bacterial communities using both a culture-dependent (MALDI-TOF) and culture-independent (NGS) methods. Environmental specimens were obtained from both freshwater and seawater. Water samples were also analyzed by both pyrosequencing and MiSeq sequencing, in order to select one NGS platform which could analyze comparatively more diverse microbiota. Bacterial distribution analyzed with MALDI-TOF showed no difference between the microbiota of freshwater and seawater, whereas the results analyzed with NGS distinguished between the two. The diversity indexes of MiSeq sequencing were higher than for Pyrosequencing. This indicated that MiSeq sequencing is capable of analyzing a comparatively wider diversity of bacteria. The genus of Flavobacterium and Planktophila were identified as being unique to freshwater, whereas EU801223 and OM43 were found in the seawater. Difference between the bacterial composition of the freshwater and seawater environments was identified by MiSeq sequencing analysis.

• 한국환경농학회지
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• 제39권1호
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• pp.26-35
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• 2020

BACKGROUND: Microbes that govern a unique biochemical process of oxidizing ammonia into dinitrogen gas, such as anaerobic ammonium oxidation (anammox) have been reported to play a pivotal role in agricultural soils and in oceanic environments. However, limited information for anammox bacterial abundance and distribution in the terrestrial habitats has been known. METHODS AND RESULTS: Phylogenetic and next-generation sequencing analyses of bacterial 16S rRNA gene were performed to examine potential anammox bacteria in paddy soils. Through clone libraries constructed by using the anammox bacteria-specific primers, some clones showed sequence similarities with Planctomycetes (87% to 99%) and anammox bacteria (94% to 95%). Microbial community analysis for the paddy soils by using Illumina Miseq sequencing of 16S rRNA gene at phylum level was dominated by unclassified Bacteria at 33.2 ± 7.6%, followed by Chloroflexi at 20.4 ± 2.0% and Acidobacteria at 17.0 ± 6.5%. Planctomycetes that anammox bacteria are belonged to was 1.5% (± 0.3) on average from the two paddy soils. CONCLUSION: We suggest evidence of anammox bacteria in the paddy soil. In addition to the relatively well-known microbial processes for nitrogen-cycle, anammox can be a potential contributor on the cycle in terrestrial environments such as paddy soils.

• Animal Systematics, Evolution and Diversity
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• 제36권4호
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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.

• 보존과학회지
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• 제37권4호
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• pp.357-361
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• 2021

In order to determine the changes in microbial community due to termites, soil microorganisms surrounding the termites were investigated. First, bacterial communities from soil with termites collected at Seonamsa temple, Suncheon city, Korea were compared by next-generation sequencing (NGS, Illumina Miseq). The bacterial composition of soil from Daeungjeon without termites and the soil from Josadang, Palsangjeon, and Samjeon with termites were compared. Next, the bacterial composition of these soils was also compared with that of humus soil cultured with termites. A total high-quality sequences of 71,942 and 72,429 reads were identified in Seonamsa temple's soil and humus soil, respectively. The dominant phyla in the collected Seonamsa temple's soil were Proteobacteria (27%), Firmicutes (24%) and Actinobacteria (21%), whereas those in the humus soil were Bacteriodetes (56%) and Proteobacteria (37%). Using a two-dimensional plot to explain the principal coordinate analysis of operational taxonomic unit compositions of the soil samples, it was confirmed that the samples were divided into soil with and without termites, and it was especially confirmed that the Proteobacteria phylum was increased in humus soil with termites than in humus soil without termites.

• 한국환경농학회지
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• 제39권1호
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• pp.1-9
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• 2020

본 연구에서는 일천궁의 연작재배에 따른 토양 이화학성 및 토양세균군집을 비교 분석하기 위해 천궁 중에서 재배 빈도수가 높은 일천궁의 초작 및 연작 재배지를 선정하여 토양 이화학성 및 토양세균군집 특성을 분석하고, 토양 이화학성 및 토양세균군집 간의 상관관계를 구명하고자 하였다. 토양 이화학성은 농촌진흥청 토양분석법을 이용하였고, 토양세균군집 분석은 Illumina Miseq sequencing system을 이용하여 상대적 빈도수 및 주좌표 분석을 하였다. 토양 이화학성과 토양세균군집 간의 상관관계는 DISTLM과 Spearman's 상관관계 분석을 이용하였다. 일천궁 재배지의 토양세균군집은 일천의 재배법에 따라 뚜렷하게 2개의 cluster로 군집화를 이루었고, 초작 및 연작 재배지 모두 Proteobacteria와 Alphaproteobacteria가 우점종으로 나타났다. 주좌표 분석과 DISTLM 분석에서는 일천궁 재배지의 토양 pH와 Ca이 토양세균의 군집화에 유의적으로 영향을 주고 있음을 확인하였고, Spearman's 상관관계 분석을 통해 일천궁의 재배법에 따라 유의적인 차이를 보였던 Acinobacteria와 Acidobacteria의 상대적 빈도수는 토양 pH, Ca 함량과 유의적인 상관관계를 보이는 것으로 나타났다. 본 연구는 일천궁의 재배법에 따른 토양 이화학성 및 토양세균군집의 변화와 상관관계를 구명하는데 중요한 자료가 될 것으로 판단된다. 또한, 토양 이화학성과 토양세균군집의 변화에 따른 일천궁 재배지의 연작장해 원인을 구명하는데 있어 도움을 줄 수 있을 것이다. 향후 본 연구를 바탕으로 향후 일천궁의 재배법에 따른 곰팡이(fungi)의 군집과 병원성 미생물군집의 변화를 확인하고 토양 이화학성과의 상관관계를 분석할 수 있다면 일천궁의 연작장해 원인을 명확하게 구명하는데 도움을 줄 수 있을 것으로 사료된다.

• 한국환경농학회지
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• 제39권3호
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• pp.178-187
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• 2020

BACKGROUND: Spent coffee grounds are the most valuable resource for agriculture and industry. However, it is almost thrown untreated into landfills or incineration. Composting is an efficient process for converting spent coffee to fertilizer. METHODS AND RESULTS: Composting was conducted in the compost pile (40 ㎥) equipped with a forced aeration system. Physical and chemical properties containing temperature, pH, electrical conductivity, and moisture were measured through the composting period. Moreover, biological changes were examined for the composting phase using Illumina Miseq sequencing of the 16S rRNA gene. We found 7-14 phyla comprising 250-716 species from a variety phase of compost. During the composting period, Firmicutes were dominated, followed by Actinobacteria and Proteobacteria. CONCLUSION: The result indicated that the use of spent coffee improved the quality of organic fertilizer and changed the microbial communities, unique to the thermal composting stage, which could enhance the composting process. These findings suggest that spent coffee composted material can provide a significant amount of nutrients, thereby supporting plant growth.

• Mycobiology
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• 제48권5호
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• pp.364-372
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• 2020

To improve our understanding of the relationship between soil higher fungi (belonging to Ascomycota and Basidiomycota) and Abies koreana, we surveyed A. koreana soil fungal communities in a forest in Mt. Halla, Jeju Island, Korea by next-generation sequencing (Illumina Miseq). To confirm the soil higher fungal communities, we collected two types of soils from a defined plot: soils with dead (AKDTs) and living A. koreana (AKLTs), respectively. Soil fungi were classified into 2 phyla, 19 classes, 64 orders, 133 families, 195 genera, and 229 OTUs (895,705 sequence reads). Nonmetric multidimensional scaling (NMDS) showed significantly different soil higher fungal communities between AKDTs and AKLTs (p < .05). In addition, the saprophyte composition was significantly affected by A. koreana status (p < .05). The proportion of the mycorrhizal Clavulina spp. was different between soils with AKDTs and AKLTs, suggesting that Clavulina spp. may be a crucial soil fungal species influencing A. koreana. This study will lead to a better understanding of the ecological status of A. koreana in Mt. Halla. In addition, this study could be useful for the conservation and management of A. koreana habitats.

• Journal of Animal Science and Technology
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• 제65권4호
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• pp.856-864
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• 2023

There are a variety of microorganisms in the animal intestine, and it has been known that they play important roles in the host such as suppression of potentially pathogenic microorganisms, modulation of the gut immunity. In addition, the gut microbiota and the livestock growth performance have long been known to be related. Therefore, we evaluated the interrelation between the growth performance and the gut microbiome of the pigs from 3 different farms, with pigs of varied ages ready to be supplied to the market. When pigs reached average market weight of 118 kg, the average age of pigs in three different farms were < 180 days, about 190 days, and > 200 days, respectively. Fecal samples were collected from pigs of age of 70 days, 100 days, 130 days, and 160 days. The output data of the 16S rRNA gene sequencing by the Illumina Miseq platform was filtered and analyzed using Quantitative Insights into Microbial Ecology (QIIME)2, and the statistical analysis was performed using Statistical Analysis of Metagenomic Profiles (STAMP). The results of this study showed that the gut microbial communities shifted as pigs aged along with significant difference in the relative abundance of different phyla and genera in different age groups of pigs from each farm. Even though, there was no statistical differences among groups in terms of Chao1, the number of observed operational taxonomic units (OTUs), and the Shannon index, our results showed higher abundances of Bifidobacterium, Clostridium and Lactobacillus in the feces of pigs with rapid growth rate. These results will help us to elucidate important gut microbiota that can affect the growth performance of pigs.

• Asian-Australasian Journal of Animal Sciences
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• 제31권9호
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• pp.1464-1473
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• 2018

Objective: To describe in-depth sequencing, the bacterial community diversity and its succession during ensiling of whole-plant maize and subsequent exposure to air. Methods: The microbial community dynamics of fermented whole-plant maize for 60 days (sampled on day 5, 10, 20, 40, 60) and subsequent aerobic exposure (sampled on day 63 after exposure to air for 3 days) were explored using Illumina Miseq sequence platform. Results: A total of 227,220 effective reads were obtained. At the genus level, there were 12 genera with relative abundance >1%, Lactobacillus, Klebsiella, Sporolactobacillus, Norank-c-cyanobacteria, Pantoea, Pediococcus, Rahnella, Sphingomonas, Serratia, Chryseobacterium, Sphingobacterium, and Lactococcus. Lactobacillus consistently dominated the bacterial communities with relative abundance from 49.56% to 64.17% during the ensiling process. Klebsiella was also an important succession bacterium with a decrease tendency from 15.20% to 6.41% during the ensiling process. The genus Sporolactobacillus appeared in late-ensiling stages with 7.70% abundance on day 40 and 5.32% on day 60. After aerobic exposure, the Lactobacillus decreased its abundance from 63.2% on day 60 to 45.03% on d 63, and Klebsiella from 5.51% to 5.64%, while Sporolactobacillus greatly increased its abundance to 28.15%. These bacterial genera belong to 5 phyla: Firmicutes (relative abundance: 56.38% to 78.43%) was dominant, others were Proteobacteria, Bacteroidetes, Cyanobacteria, and Actinobacteria. The bacterial communities clearly clustered into early-ensiling (d 5), medium-ensiling (d 10, d 20), late-ensiling (d 40, d 60), and aerobic exposure (d 63) clusters, with early- and late-ensiling communities more like each other than to the aerobic exposure communities. Conclusion: High-throughput sequencing based on 16S rRNA genes proved to be a useful method to explore bacterial communities of silage. The results indicated that the bacterial communities varied during fermentation and more dramatically during aerobic exposure. The study is valuable for understanding the mechanism of population change and the relationship between bacteria and ensilage characteristics.

• 한국어류학회지
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• 제35권4호
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• pp.224-235
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• 2023

우리나라는 전 세계에서 유일한 분단국가로 군사분계선을 가지고 있다. 군사분계선을 기준으로 비무장지대 및 민간인통제구역이 설정되었고, 국가보안 및 안전을 위해 출입이 통제되고 있어, 우리나라 다른 지역에 비해 생태계 교란이 비교적 적은 지역이다. 하지만 유실지뢰 및 불발탄들로 인해 안전이 위협받음에 따라 생태계 연구에 많은 제한사항이 발생하기 때문에, 연구를 보완 및 대체하기 위해 eDNA 분석법을 활용하여 민간인통제구역 내부에 존재하는 평화의 길 세 지점에서 직접조사와 eDNA를 이용한 간접조사를 실시하고 비교했다. 평화의 길 인제노선, 양구노선, 화천노선 세 지점에서 2022년 5, 7, 9월에 직접조사와 eDNA 시료 채취를 진행했으며, 시료에서 채취한 유전자를 증폭한 후 library를 제작하여 Miseq를 수행하고 ASV를 생성하여 간접조사를 완료했다. 이후, 직접조사 결과와 비교했다. 그 결과 양구-1 지점에서 관찰된 2종 모두 eDNA가 검출되었으며, 양구-2 지점에서 관찰된 4종 중 2종의 eDNA가 검출되었다. 화천-1에서 관찰된 1종 중 1종의 eDNA가 검출되었고, 화천-2에서 12종 중 7종, 화천-3에서는 6종 중 4종, 화천-4에서 관찰된 1종 모두 eDNA가 검출되어 직접조사 결과 확인된 종의 약 69%의 어류의 eDNA가 검출된 간접조사 결과를 확인했다. 대륙종개, 메기 등 일부 어류가 오동정된 상태로 NCBI에 유전정보가 등재된 것은 아닌지 확인이 필요하고, 다른 서열부를 이용한 마커 개발 연구가 필요할 것으로 생각되며, 위험성 때문에 조사가 제한적인 CCZ 지역 어류 연구의 보완책으로써 적합할 것으로 생각된다.