• Genomics & Informatics
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• v.6 no.2
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• pp.87-90
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• 2008

During the last four years, the pyrosequencing-based 454 platform has rapidly displaced the traditional Sanger sequencing method due to its high throughput and cost effectiveness. Meanwhile, the Sanger sequencing methodology still provides the longest reads, and paired-end sequencing that is based on that chemistry offers an opportunity to ensure accurate assembly results. In this report, we describe an optimized approach for hybrid de novo genome assembly using pyrosequencing data and varying amounts of Sanger-type reads. 454 platform-derived contigs can be used as single non-breakable virtual reads or converted to simpler contigs that consist of editable, overlapping pseudoreads. These modified contigs maintain their integrity at the first jumpstarting assembly stage and are edited by fragmenting and rejoining. Pre-existing assembly software then can be applied for mixed assembly with 454-derived data and Sanger reads. An effective method for identifying genomic differences between reference and sample sequences in whole-genome resequencing procedures also is suggested.

• Korean Journal of Microbiology
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• v.49 no.3
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• pp.237-244
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• 2013

Bacterial diversities in the guts of sea cucumbers (Apostichopus japonicus) and shrimps (Litopenaeus vannamei) were investigated using barcoded or tag-encoded 454 pyrosequencing of 16S rRNA genes. In sea cucumbers, most of sequences were related to two genera, the genus Propionigenium in the phylum Fusobacteria and an unclassified genus in the family Flavobacteriaceae of phylum Bacteroidetes. Shrimps showed various kinds of genera including Lactococcus, Leuconostoc, Prochlorococcus, and Vibrio as well as the unclassified genera in the families, Flavobacteriaceae, Rhodobacteraceae, Desulfobulbaceae, and Helicobacteraceae and in the order Mycoplasmatales. Unclassified genera containing environmental sequences only are more than half of genera from sea cucumbers and shrimps. Sea cucumbers and shrimps could be unexplored sources of novel microbes and the bacterial diversity of them was revealed by high throughput 454 pyrosequencing.

• Korean Journal of Microbiology
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• v.47 no.3
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• pp.209-217
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• 2011

Archaeal communities were investigated using 454 pyrosequencing technology based on 16S rRNA gene in 11 samples collected from six different full-scale anaerobic digesters. Observed operational taxonomic units (OTUs) estimated from the archaeal 16S rRNA gene sequences were 13-55 OTUs (3% cutoff) which was corresponded to 29-89% of Chao1 richness estimates. In the anaerobic digesters there were archaeal sequences within the orders Thermoproteales, Thermoplasmatales, Desulfurococcales as well as within the orders Methanomicrobiales, Methanobacteriales, Methanococcales, Methanosarcinales, and Methanocellales, which are known to produce methane. Among these orders, Methanococcales known to produce methane using hydrogen was the predominant taxon and constituted 51.8-99.7% of total sequences. All samples showed a very similar community structure (Pearson correlation coefficient=0.99) except for one sample based on a heat map analysis. In addition, canonical correspondence analysis correlating archaeal communities to the environmental variables demonstrated that digester temperature and total solids removal rate were the two important explanatory variables. Overall results suggested that environmental and operational variables of anaerobic digester are important factors determining archaeal diversity and community structure.

• Journal of Microbiology and Biotechnology
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• v.24 no.3
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• pp.313-323
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• 2014

Significant alteration in the microbial community can occur across reclamation areas suffering subsidence from mining. A reclamation site undergoing fertilization practices and an adjacent coal-excavated subsidence site (sites A and B, respectively) were examined to characterize the bacterial diversity using 454 high-throughput 16S rDNA sequencing. The dominant taxonomic groups in both the sites were Proteobacteria, Acidobacteria, Bacteroidetes, Betaproteobacteria, Actinobacteria, Gammaproteobacteria, Alphaproteobacteria, Deltaproteobacteria, Chloroflexi, and Firmicutes. However, the bacterial communities' abundance, diversity, and composition differed significantly between the sites. Site A presented higher bacterial diversity and more complex community structures than site B. The majority of sequences related to Proteobacteria, Gemmatimonadetes, Chloroflexi, Nitrospirae, Firmicutes, Betaproteobacteria, Deltaproteobacteria, and Anaerolineae were from site A; whereas those related to Actinobacteria, Planctomycetes, Bacteroidetes, Verrucomicrobia, Gammaproteobacteria, Nitriliruptoria, Alphaproteobacteria, and Phycisphaerae originated from site B. The distribution of some bacterial groups and subgroups in the two sites correlated with soil properties and vegetation due to reclamation practice. Site A exhibited enriched bacterial community, soil organic matter (SOM), and total nitrogen (TN), suggesting the presence of relatively diverse microorganisms. SOM and TN were important factors shaping the underlying microbial communities. Furthermore, the specific plant functional group (legumes) was also an important factor influencing soil microbial community composition. Thus, the effectiveness of 454 pyrosequencing in analyzing soil bacterial diversity was validated and an association between land ecological system restoration, mostly mediated by microbial communities, and an improvement in soil properties in coal-mining reclamation areas was suggested.

• Journal of Microbiology and Biotechnology
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• v.26 no.1
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• pp.110-119
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• 2016

High-solid anaerobic digestion of sewage sludge achieves highly efficient volatile solid reduction, and production of volatile fatty acid (VFA) and methane compared with conventional low-solid anaerobic digestion. In this study, the potential mechanisms of the better performance in high-solid anaerobic digestion of sewage sludge were investigated by using 454 high-throughput pyrosequencing and real-time PCR to analyze the microbial characteristics in sewage sludge fermentation reactors. The results obtained by 454 highthroughput pyrosequencing revealed that the phyla Chloroflexi, Bacteroidetes, and Firmicutes were the dominant functional microorganisms in high-solid and low-solid anaerobic systems. Meanwhile, the real-time PCR assays showed that high-solid anaerobic digestion significantly increased the number of total bacteria, which enhanced the hydrolysis and acidification of sewage sludge. Further study indicated that the number of total archaea (dominated by Methanosarcina) in a high-solid anaerobic fermentation reactor was also higher than that in a low-solid reactor, resulting in higher VFA consumption and methane production. Hence, the increased key bacteria and methanogenic archaea involved in sewage sludge hydrolysis, acidification, and methanogenesis resulted in the better performance of high-solid anaerobic sewage sludge fermentation.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.1
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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.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.364-364
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• 2017

Saemangeum reclaimed land is a part of Saemangeum Development Project. Most of the persistent problems of Saemangeum reclaimed land remain to be related to soil salinity. Soil salinity is a major abiotic factor related to microbial community structure and also fungi have been reported to be more sensitive to salinity stress than bacteria. The aim of this study was conducted to investigate the effect of soil salinity levels on the microbial communities in Saemangeum reclaimed land using 454 pyrosequencing analysis. Soil samples was collected from 12 sites of in Saemangeum reclaimed land. For pyrosequencing, 27F/518R (bacteria) and ITS3/ITS4 (fungi) primers were used containing the Roche 454 pyrosequencing adaptor-key-linker (underlined) and unique barcodes (X). Pyrosequencing was performed by Chun's Lab (Seoul, Korea) using the standard shotgun sequencing reagents and a 454 GS FLX Titanium sequencing System (Roche, Inc.). In the soil samples, Proteobacteria (bacteria) and Ascomycota (fungi) shows the highest relative abundance in all the soil sample sites. Proteobacteria, Bacteroidetes, Plantomycetes, Gemmatimonadetes and Parcubacteria were shown to have significantly higher abundance in high salinity level soils than low salinity level soils, while Acidobacteria and Nitrospirae has significantly higher relative abundance in low salinity level soils. The abundance of fungal, Ascomycota has the highest relative abundance in soil samples, followed by Basidiomycota, Chlorophyta, Zygomycota and Chytridiomycota. Basidiomycota, Zygomycota, Glomeromycota and Cerozoa were show significantly higher relative abundance in low salinity level soils. The principal coordinate analysis (PCoA) and correlation analysis shown to salinity-related soil parameters such as ECe, Na+, SAR and EPS were affected to bacterial and fungal community structure. Proteobacteria, Bacteroidetes, Plantomycetes exhibited significantly positive correlation with soil salinity, while Acidobacteria exhibited significantly negative correlation. In the case of fungal community, Basidiomycota and Zygomycota were seen show significantly negative correlation with salinity related soil parameters. These results suggest that provide understanding effect of soil salinity on microbial community structure and correlation of microbial community with soil parameters in Saemangeum reclaimed land.

• Journal of Dairy Science and Biotechnology
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• v.36 no.2
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• pp.125-131
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• 2018

The present study aimed to investigate the microbial diversity in Gouda cheese within the four months of ripening, via next-generation sequencing (NGS). Lactococcus (96.03%), and Leuconostoc (3.83%), used as starter cultures, constituted the majority of bacteria upon 454 pyrosequencing based on 16S rDNA sequences. However, no drastic differences were observed among other populations between the center and the surface portions of Gouda cheese during ripening. Although the proportion of subdominant species was <1%, slight differences in bacterial populations were observed in both the center and the surface portions. Taken together, our results suggest that environmental and processing variables of cheese manufacturing including pasteurization, starter, ripening conditions are important factors influencing the bacterial diversity in cheese and they can be used to alter nutrient profiles and metabolism and the flavor during ripening.

• Korean Journal of Soil Science and Fertilizer
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• v.45 no.6
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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 Life Science
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• v.31 no.4
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• pp.377-384
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• 2021

Researching the soil fungal community is important to understand the interaction between fungi and living plants. However, too few studies have examined the soil fungal community and their interactions with plants. Rhododendron mucronulatum, commonly known as Korean rosebay, is an important forest resource that has aesthetic, ecological, and potential pharmacological values. We used a pyrosequencing method to analyze the characteristics of fungal communities from R. mucronulatum soil samples from Biseul mountain county park, which is one of the famous places for large R. mucronulatum colonies in South Korea. We collected soil core samples in February and August at three sites in the Biseul Mountain County Park, taking into consideration the regional and seasonal conditions. We obtained 454,157 validated reads after pyrosequencing all six samples. The fungal communities from the first observation spot in August had the richest species diversity among the samples. Basidiomycota, Ascomycota, and Mortierellomycota were major phyla in the samples. Agaricales_f, Mortierellaceae, and Clavariaceae were major families in the samples. The genus Mortierella was the most dominant in all six samples. Overall, 19 genera could be associated with R. mucronulatum. Sample 1 had 109 genera in sample 1, sample 2 had 111 genera, and sample 3 had 112 genera that were uniquely identified. The samples collected in August had 28 identified genera, that existed only in summer samples, indicating a weather effect. This study can be used as basic research to understand the relationship between soil fungi and plants.