• 한국토양비료학회지
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• 제45권6호
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• pp.1073-1085
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• 2012

Soil microbial communities are immensely diverse and complex with respect to species richness and community size. These communities play essential roles in agricultural soil because they are responsible for most of the nutrient cycles in the soil and influence the plant diversity and productivity. However, the majority of these microbes remain uncharacterized because of poor culturability. Next-generation sequencing techniques have revolutionized many areas of biology by providing cheaper and faster alternatives to Sanger sequencing. Among them, amplicon pyrosequencing is a powerful tool developed by 454 Life Sciences for assessing the diversity of complex microbial communities by sequencing PCR products or amplicons. This review summarizes the current opinions in amplicon sequencing of soil microbial communities, and provides practical guidance and advice on sequence quality control, aligning, clustering, OTU- and taxon-based analysis. The last section of this article includes a few representative studies conducted using amplicon pyrosequencing.

• Journal of Microbiology and Biotechnology
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• 제25권1호
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• pp.1-10
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• 2015

The bacterial diversity of 10 marine sponges belonging to the species Cliona celata, an unidentified Cliona species, Haliclona cinerea, Halichondria okadai, Hymeniacidon sinapium, Lissodendoryx isodictyalis, Penares incrustans, Spirastrella abata, and Spirastrella panis collected from Jeju Island and Chuja Island was investigated using amplicon pyrosequencing of the 16S rRNA genes. The microbial diversity of these sponges has as of yet rarely or never been investigated. All sponges, except Cliona celata, Lissodendoryx isodictyalis, and Penares incrustans, showed simple bacterial diversity, in which one or two bacterial OTUs occupied more than 50% of the pyrosequencing reads and their OTU rank abundance curves saturated quickly. Most of the predominant OTUs belonged to Alpha-, Beta-, or Gammaproteobacteria. Some of the OTUs from the sponges with low diversity were distantly (88%~89%) or moderately (93%~97%) related to known sequences in the GenBank nucleotide database. Phylogenetic analysis showed that many of the representative sequences of the OTUs were related to the sequences originating from sponges and corals, and formed sponge-specific or -related clades. The marine sponges investigated herein harbored unexplored bacterial diversity, and further studies should be done to understand the microbes present in sponges.

• ALGAE
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• 제30권3호
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• pp.223-232
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• 2015

More than 20 and 10 clades / ecotypes of Synechococcus and Prochlorococcus, respectively, have been identified in various oceanic regions. However, their diversity has yet to be thoroughly studied in the northwest Pacific Ocean. Further, spatial distribution of Synechococcus clades in the oligotrophic oceans has been scarcely characterized. To elucidate picocyanobacterial lineage distribution in the northwest Pacific Ocean, 16S-23S internal transcribed spacer sequences of picocyanobacteria were sequenced by barcoded amplicon pyrosequencing method. Additional pyrosequencing library using a primer specific for the Synechococcus subcluster-5.1 was constructed to thoroughly understand Synechococcus diversity in the oligotrophic oceans. In warm pool area, Prochlorococcus was predominant and showed a distinct depthpartitioning between HLII and LL ecotypes. Despite low abundances, diverse Synechococcus clades appeared in the oligotrophic open ocean, showing both vertical and horizontal niche partitioning. Clade II was the predominant Synechococcus clade, especially in upper euphotic depths. In shallow and middle euphotic depths, clades UC-A, III, and CRD1 were distributed broadly. However, a distinct shift in the horizontal distribution was found at ca. $20^{\circ}N$. Conversely, clades XVII and CRD2 dominated at deep euphotic depths and constituted a higher proportion than clade II. These niche-partitioning of Synechococcus clades seemed to be related with temperature, nutrient concentration as well as iron concentration.

• Asian-Australasian Journal of Animal Sciences
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• 제30권1호
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• pp.100-110
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• 2017

Objective: The gastrointestinal tract of sheep contain complex microbial communities that influence numerous aspects of the sheep's health and development. The objective of this study was to analyze the composition and diversity of the microbiota in the gastrointestinal tract sections (rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, cecum, colon, and rectum) of sheep. Methods: This analysis was performed by 454 pyrosequencing using the V3-V6 region of the 16S rRNA genes. Samples were collected from five healthy, small tailed Han sheep aged 10 months, obtained at market. The bacterial composition of sheep gastrointestinal microbiota was investigated at the phylum, class, order, family, genus, and species levels. Results: The dominant bacterial phyla in the entire gastrointestinal sections were Firmicutes, Bacteroidetes, and Proteobacteria. In the stomach, the three most dominant genera in the sheep were Prevotella, unclassified Lachnospiraceae, and Butyrivibrio. In the small intestine, the three most dominant genera in the sheep were Escherichia, unclassified Lachnospiraceae, and Ruminococcus. In the large intestine, the three most dominant genera in the sheep were Ruminococcus, unclassified Ruminococcaceae, and Prevotella. R. flavefaciens, B. fibrisolvens, and S. ruminantium were three most dominant species in the sheep gastrointestinal tract. Principal Coordinates Analysis showed that the microbial communities from each gastrointestinal section could be separated into three groups according to similarity of community composition: stomach (rumen, reticulum, omasum, and abomasum), small intestine (duodenum, jejunum, and ileum), and large intestine (cecum, colon, and rectum). Conclusion: This is the first study to characterize the entire gastrointestinal microbiota in sheep by use of 16S rRNA gene amplicon pyrosequencing, expanding our knowledge of the gastrointestinal bacterial community of sheep.

• 한국미생물·생명공학회지
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• 제47권3호
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• pp.423-433
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• 2019

Midgut microbiota is known to play a fundamental role in the biology and physiology of the agricultural pest, Spodoptera litura. This study reports the difference in the larval midgut microbiota of field-caught and laboratory-reared populations of S. litura by performing 16S rDNA amplicon pyrosequencing. Field populations for the study were collected from castor crops, whereas laboratory-reared larvae were fed on a regular chickpea based diet. In total, 23 bacterial phylotypes were observed from both laboratory-reared and field-caught caterpillars. Fisher's exact test with Storey's FDR multiple test correction demonstrated that bacterial genus, Clostridium was significantly abundant (p < 0.05) in field-caught larvae of S. litura as compared to that in the laboratory-reared larvae. Similarly, bacterial genera, such as Bradyrhizobium, Burkholderia, and Fibrisoma were identified (p < 0.05) predominantly in the laboratory-reared population. The Bray-Curtis dissimilarity matrix depicted a value of 0.986, which exhibited the maximum deviation between the midgut microbiota of the laboratory-reared and field-caught populations. No significant yeast diversity was seen in the laboratory-reared caterpillars. However, two yeast strains, namely Candida rugosa and Cyberlindnera fabianii were identified by PCR amplification and molecular cloning of the internal transcribed space region in the field-caught caterpillars. These results emphasize the differential colonization of gut residents based on environmental factors and diet.

• Restorative Dentistry and Endodontics
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• 제36권6호
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• pp.498-505
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• 2011

연구목적: 본 연구는 원발성 치근단 치주염(primary apical periodontitis)을 갖는 치아에서 임상증상 유무에 따른 미생물 군집의 차이를 GS FLX Titanium pyrosequencing을 이용하여 species level까지 분석하였다. 연구 재료 및 방법: 원발성 치근단 치주염을 갖는 6개의 표본에서 pysequencing을 시행하였다. 중합효소 연쇄반응(PCR)에 의해 얻어진 small-subunit ribosomal RNA의 초가변 영역(hypervariable region)의 amplicon을 이용하여 GS FLX Titanium pyrosequencing 을 시행하였다. 결과: 평균적으로 무증상군 및 증상군에서 각각 10,639 및 45,455개의 16S rRNA sequence을 얻었으며 평균길이는 440bases였다. Ribosomal Database Project Classifier을 이용한 분석결과 142종의 genera 및 13종의 phylum 수준에서의 세균종을 검출하였다. 검출된 13개의 phyla 가운데 Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, Spirochetes, and Synergistetes 종이 상대적으로 호발하였으며, genus 수준에서는 Pyramidobacter, Streptococcus, Leptotrichia이 무증상의 근관의 50%를 차지하였으며, Neisseria, Propionibacterium, Tessaracoccus 균종은 증상이 있는 근관의 69%를 차지하였다. Operational taxonomic units (3%)로 나눈 결과 증상이 없는 치아에서 450개, 증상이 있는 치아에서 1,997개의 species가 발견되었다. 증상이 있는 치아에서 통계적으로 유의성 있게 많은 수의 세균이 검출되었다(p < 0.05). 결론: GS FLX Titanium Pyrosequencing 기법을 통해 원발성 감염근관에서 이전에 검출하지 못했던 다양한 근관내 분포세균을 검출할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제25권6호
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• pp.765-770
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• 2015

Recent sequencing technology development has revolutionized fields of microbial ecology. MiSeq-based microbial community analysis allows us to sequence more than a few hundred samples at a time, which is far more cost-effective than pyrosequencing. The approach, however, has not been preferably used owing to computational difficulties of processing huge amounts of data as well as known Illumina-derived artefact problems with amplicon sequencing. The choice of assembly software to take advantage of paired-end sequencing and methods to remove Illumina artefacts sequences are discussed. The protocol we suggest not only removed erroneous reads, but also dramatically reduced computational workload, which allows even a typical desktop computer to process a huge amount of sequence data generated with Illumina sequencers. We also developed a Web interface (http://biotech.jejunu.ac.kr/ ~abl/16s/) that allows users to conduct fastq-merging and mothur batch creation. The study presented here should provide technical advantages and supports in applying MiSeq-based microbial community analysis.

• Ocean and Polar Research
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• 제34권1호
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• pp.19-28
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• 2012

In order to understand the spatial distribution of picocyanobacterial diversity during the summer in the northern East China Sea (ECS), their abundance and genetic diversity were investigated using flow cytometry and barcoded amplicon pyrosequencing of 16S-23S internal transcribed spacer sequences. Synechococcus abundance was high, with a range of $0.2{\times}10^5$ to $1.8{\times}10^5$ cells $ml^{-1}$. However, Prochlorococcus were found only in the eastern part of the studied area, showing a marked variation among stations [range of n.d. (not detected) to $3.3{\times}10^4$ cells $ml^{-1}$]. Eleven Synechococcus clades and five Prochlorococcus ecotypes were found to have a proportion higher than 1% among picocyanobacterial sequences, indicating high picocyanobacterial diversity in the ECS. The picocyanobacterial compositions were markedly different among stations, as well as among depths. Inflow of the Tsushima Warm Current and Changjiang diluted water was of primary importance in determining picocyanobacterial lineage diversity in the studied area. In addition, light intensity and nutrient conditions also appeared to be important in the vertical and horizontal distribution of picocyanobacterial diversity.

• 한국유기농업학회지
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• 제27권4호
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• pp.479-499
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• 2019

본 연구는 제주지역 무 주산지에서 재배 방법(유기 vs. 관행)과 토양 종류(화산회토 vs. 비화산회토)에 따른 토양의 화학적 특성, 미생물 활성 그리고 미생물 군집 조성을 분석하고 요인간 연관성을 구명하기 위하여 수행하였다. 전반적으로 유기와 관행의 재배 방식에 따른 토양 화학성은 처리간 뚜렷한 경향을 보이지는 않았으나 토양 미생물체량, 효소 활성, 종 풍부도와 다양성 그리고 미생물 군집 분포 등은 유의한 차이를 보였다. 반면에 토양 종류에 따른 화학성과 미생물 군집 분포 등 미생물학적 특성은 뚜렷한 차이를 보였다. 특히 유기재배 토양에서 관행 대비 토양의 세균, 방선균 및 사상균 그리고 미생물체량이 증가하였으며, Org-NA 토양에서 탈수소효소 활성, 종 풍부도(Chao 1) 그리고 종 다양성(Phyrogenetic diversity) 지수가 가장 높았다. 무 재배 토양에 분포하고 있는 주요 세균 문은 Proteobacteria, Acidobacteria, Chloroflexi, Firmicutes 그리고 Actinobacteria 등 5종이었으며 재배 방법 및 토양 종류에 관계없이 Proteobacteria 문이 화산회토에서 25.9%, 비화산회토에서 21.9~24.9%로 가장 높은 분포를 보였다. 그리고 대체로 화산회토와 비화산회토 토양 종류별로 유사한 군집 조성을 보였으며, 화산회토에서는 재배 방법별 주요 문의 군집 조성은 큰 차이가 없었으나 비화산회토에서는 차이를 보였다. 특히, Firmicutes는 Org-NA 토양에서 21.0%, Acidobacteria는 Con-A에서 21.6%로 가장 높은 분포를 보였는데 대체로 화산회토와 관행재배 토양에서 높은 경향을 보였다. 또한 재배 방법 및 토양 종류별 미생물 군집을 대표하는 바이오마커를 찾기 위하여 LEfSe 분석을 실시한 결과, Firmicutes 문의 분포가 비화산회토와 유기재배 토양에서 유의하게 증가하였다. 그리고 토양 화학성 중에서 총유기탄소 함량, 유효인산 그리고 치환성칼륨 함량은 Firmicutes 등 주요 세균 문과 유의한 상관관계를 보였다.