- Improved pipeline for reducing erroneous identification by 16S rRNA sequences using the Illumina MiSeq platform
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Yoon-Seong Jeon , Sang-Cheol Park , Jeongmin Lim , Jongsik Chun , Bong-Soo Kim
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J. Microbiol. 2015;53(1):60-69. Published online January 4, 2015
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DOI: https://doi.org/10.1007/s12275-015-4601-y
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Abstract
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The cost of DNA sequencing has decreased due to advancements
in Next Generation Sequencing. The number of sequences
obtained from the Illumina platform is large, use of
this platform can reduce costs more than the 454 pyrosequencer.
However, the Illumina platform has other challenges,
including bioinformatics analysis of large numbers
of sequences and the need to reduce erroneous nucleotides
generated at the 3-ends of the sequences. These erroneous
sequences can lead to errors in analysis of microbial communities.
Therefore, correction of these erroneous sequences
is necessary for accurate taxonomic identification. Several
studies that have used the Illumina platform to perform metagenomic
analyses proposed curating pipelines to increase
accuracy. In this study, we evaluated the likelihood of obtaining
an erroneous microbial composition using the MiSeq
250 bp paired sequence platform and improved the pipeline
to reduce erroneous identifications. We compared different
sequencing conditions by varying the percentage of control
phiX added, the concentration of the sequencing library, and
the 16S rRNA gene target region using a mock community
sample composed of known sequences. Our recommended
method corrected erroneous nucleotides and improved identification
accuracy. Overall, 99.5% of the total reads shared
95% similarity with the corresponding template sequences
and 93.6% of the total reads shared over 97% similarity. This
indicated that the MiSeq platform can be used to analyze microbial
communities at the genus level with high accuracy.
The improved analysis method recommended in this study
can be applied to amplicon studies in various environments
using high-throughput reads generated on the MiSeq platform.
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- Assessment of Soil Fungal Communities Using Pyrosequencing
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Young Woon Lim , Byung Kwon Kim , Changmu Kim , Hack Sung Jung , Bong-Soo Kim , Jae-Hak Lee , Jongsik Chun
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J. Microbiol. 2010;48(3):284-289. Published online June 23, 2010
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DOI: https://doi.org/10.1007/s12275-010-9369-5
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681
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Abstract
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Pyrosequencing, a non-electrophoretic method of DNA sequencing, was used to investigate the extensive fungal community in soils of three islands in the Yellow Sea of Korea, between Korea and China. Pyrosequencing was carried out on amplicons derived from the 5′ region of 18S rDNA. A total of 10,166 reads were obtained, with an average length of 103 bp. The maximum number of fungal phylotypes in soil predicted at 99% similarity was 3,334. The maximum numbers of phylotypes predicted at 97% and 95% similarities were 736 and 286, respectively. Through phylogenetic assignment using BLASTN, a total of 372 tentative taxa
were identified. The majority of true fungal sequences recovered in this study belonged to the Ascomycota (182 tentative taxa in 2,708 reads) and Basidiomycota (172 tentative taxa in 6,837 reads). The predominant species of Ascomycota detected have been described as lichen-forming fungi, litter/wood decomposers, plant
parasites, endophytes, and saprotrophs: Peltigera neopolydactyla (Lecanoromycetes), Paecilomyces sp. (Sordariomycetes), Phacopsis huuskonenii (Lecanoromycetes), and Raffaelea hennebertii (mitosporic Ascomycota). The majority of sequences in the Basidiomycota matched ectomycorrhizal and wood rotting fungi, including species of the Agaricales and Aphyllophorales, respectively. A high number of sequences in the Thelephorales, Boletales, Stereales, Hymenochaetales, and Ceratobasidiomycetes were also detected. By
applying high-throughput pyrosequencing, we observed a high diversity of soil fungi and found evidence that pyrosequencing is a reliable technique for investigating fungal communities in soils.
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- Rapid Phylogenetic Dissection of Prokaryotic Community Structure in Tidal Flat Using Pyrosequencing
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Bong-Soo Kim , Byung Kwon Kim , Jae-Hak Lee , Myungjin Kim , Young Woon Lim , Jongsik Chun
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J. Microbiol. 2008;46(4):357-363. Published online August 31, 2008
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DOI: https://doi.org/10.1007/s12275-008-0071-9
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Dissection of prokaryotic community structure is prerequisite to understand their ecological roles. Various methods are available for such a purpose which amplification and sequencing of 16S rRNA genes gained its popularity. However, conventional methods based on Sanger sequencing technique require cloning process prior to sequencing, and are expensive and labor-intensive. We investigated prokaryotic community structure in tidal flat sediments, Korea, using pyrosequencing and a subsequent automated bioinformatic pipeline for the rapid and accurate taxonomic assignment of each amplicon. The combination of pyrosequencing and bioinformatic analysis showed that bacterial and archaeal communities were more diverse than previously reported in clone library studies. Pyrosequencing analysis revealed 21 bacterial divisions and 37 candidate divisions. Proteobacteria was the most abundant division in the bacterial community, of which Gammaand Delta-Proteobacteria were the most abundant. Similarly, 4 archaeal divisions were found in tidal flat sediments. Euryarchaeota was the most abundant division in the archaeal sequences, which was further divided into 8 classes and 11 unclassified euryarchaeota groups. The system developed here provides a simple, in-depth and automated way of dissecting a prokaryotic community structure without extensive pretreatment such as cloning.
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- Archaeal Diversity in Tidal Flat Sediment as Revealed by 16S rDNA Analysis
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Bong-Soo Kim , Huyn-Myung Oh , Hojeong Kang , Jongsik Chun
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J. Microbiol. 2005;43(2):144-151.
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DOI: https://doi.org/2170 [pii]
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Abstract
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During the past ten years, Archaea have been recognized as a widespread and significant component of marine picoplankton assemblages. More recently, the presence of novel archaeal phylogenetic lineages has been discovered in coastal marine environments, freshwater lakes, polar seas, and deep-sea hydrothermal vents. Therefore, we conducted an investigation into the archaeal community existing in tidal flat sediment collected from Ganghwa Island, Korea. Phylogenetic analysis of archaeal 16S rDNA amplified directly from tidal flat sediment DNA revealed the presence of two major lineages, belonging to the Crenarchaeota (53.9%) and Euryarchaeota (46.1%) phyla. A total of 102 clones were then sequenced and analyzed by comprehensive phylogenetic analysis. The sequences determined in our samples were found to be closely related to the sequences of clones which had been previously obtained from a variety of marine environments. Archaeal clones exhibited higher similarities (83.25 - 100%) to sequences from other environments in the public database than did those (75.22 - 98.46%) of previously reported bacterial clones obtained from tidal flat sediment. The results of our study suggest that the archaeal community in tidal flat sediment is remarkably diverse.
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