- PyroTrimmer: a Software with GUI for Pre-Processing 454 Amplicon Sequences
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Jeongsu Oh , Byung Kwon Kim , Wan-Sup Cho , Soon Gyu Hong , Kyung Mo Kim
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J. Microbiol. 2012;50(5):766-769. Published online November 4, 2012
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DOI: https://doi.org/10.1007/s12275-012-2494-6
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The ultimate goal of metagenome research projects is to understand the ecological roles and physiological functions of the microbial communities in a given natural environment. The 454 pyrosequencing platform produces the longest reads among the most widely used next generation sequencing platforms. Since the relatively longer reads of the 454 platform provide more information for identification of microbial sequences, this platform is dedicated to microbial community and population studies. In order to accurately perform the downstream analysis of the 454 multiplex datasets, it is necessary to remove artificially designed sequences located at either ends of individual reads and to correct lowquality sequences. We have developed a program called Pyro-Trimmer that removes the barcodes, linkers, and primers, trims sequence regions with low quality scores, and filters out low-quality sequence reads. Although these functions have previously been implemented in other programs as well, PyroTrimmer has novelty in terms of the following features: i) more sensitive primer detection using Levenstein distance and global pairwise alignment, ii) the first stand-alone software with a graphic user interface, and iii) various options for trimming and filtering out the low-quality sequence reads. PyroTrimmer, written in JAVA, is compatible with multiple operating systems and can be downloaded free at http://pyrotrimmer.kobic.re.kr.
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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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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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- Identification and Characterization of Metagenomic Fragments from Tidal Flat Sediment
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Byung Kwon Kim , Yoon-Dong Park , Hyun-Myung Oh , Jongsik Chun
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J. Microbiol. 2009;47(4):402-410. Published online September 9, 2009
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DOI: https://doi.org/10.1007/s12275-009-0099-5
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Abstract
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Phylogenetic surveys based on cultivation-independent methods have revealed that tidal flat sediments are environments with extensive microbial diversity. Since most of prokaryotes in nature cannot be easily cultivated under general laboratory conditions, our knowledge on prokaryotic dwellers in tidal flat sediment is mainly based on the analysis of metagenomes. Microbial community analysis based on the 16S rRNA gene and other phylogenetic markers has been widely used to provide important information on the role of microorganisms, but it is basically an indirect means, compared with direct sequencing of metagenomic DNAs. In this study, we applied a sequence-based metagenomic approach to characterize uncultivated prokaryotes from tidal flat sediment. Two large-insert genomic libraries based on fosmid were constructed from tidal flat metagenomic DNA. A survey based on end-sequencing of selected fosmid clones resulted in the identification of clones containing 274 bacterial and 16 archaeal homologs in which majority were of proteobacterial origins. Two fosmid clones containing large metagenomic DNAs were completely sequenced using the shot- gun method. Both DNA inserts contained more than 20 genes encoding putative proteins which implied their ecological roles in tidal flat sediment. Phylogenetic analyses of evolutionary conserved proteins indicate that these clones are not closely related to known prokaryotes whose genome sequence is known, and genes in tidal flat may be subjected to extensive lateral gene transfer, notably between domains Bacteria and Archaea. This is the first report demonstrating that direct sequencing of metagenomic gene library is useful in underpinning the genetic makeup and functional roles of prokaryotes in tidal flat sediments.
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Citations
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- Predicting bacterial interaction outcomes from monoculture growth and supernatant assays
Désirée A Schmitz, Tobias Wechsler, Ingrid Mignot, Rolf Kümmerli ISME Communications.2024;[Epub] CrossRef - Archaeal community structure and response to ark shell bioturbation in typical intertidal mudflats, Southeast coast of China
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Lucie Bittner, Sébastien Halary, Claude Payri, Corinne Cruaud, Bruno de Reviers, Philippe Lopez, Eric Bapteste Biology Direct.2010;[Epub] CrossRef
- 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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775
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0
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55
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Abstract
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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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