• Genomics & Informatics
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• v.16 no.4
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• pp.29.1-29.5
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• 2018

Hybrid capture-based targeted sequencing is being used increasingly for genomic variant profiling in tumor patients. Unique molecular index (UMI) technology has recently been developed and helps to increase the accuracy of variant calling by minimizing polymerase chain reaction biases and sequencing errors. However, UMI-adopted targeted sequencing data analysis is slightly different from the methods for other types of omics data, and its pipeline for variant calling is still being optimized in various study groups for their own purposes. Due to this provincial usage of tools, our group built an analysis pipeline for global application to many studies of targeted sequencing generated with different methods. First, we generated hybrid capture-based data using genomic DNA extracted from tumor tissues of colorectal cancer patients. Sequencing libraries were prepared and pooled together, and an 8-plexed capture library was processed to the enrichment step before 150-bp paired-end sequencing with Illumina HiSeq series. For the analysis, we evaluated several published tools. We focused mainly on the compatibility of the input and output of each tool. Finally, our laboratory built an analysis pipeline specialized for UMI-adopted data. Through this pipeline, we were able to estimate even on-target rates and filtered consensus reads for more accurate variant calling. These results suggest the potential of our analysis pipeline in the precise examination of the quality and efficiency of conducted experiments.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.4
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• pp.1151-1158
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• 2012

High-risk human papillomavirus (HPV) genotypes are the major cause of cervical cancer. Hence, HPV genotype detection is a helpful preventive measure to combat cervical cancer. Recently, several HPV detection methods have been developed, each with different sensitivities and specificities. The objective of this study was to compare HPV high risk genotype detection by an electrochemical DNA chip system, a line probe assay (INNO-LiPA) and sequencing of the L1, E1 regions. A total of 361 cervical smears with different cytological findings were subjected to polymerase chain reaction-sequencing and electrochemical DNA chip assessment. Multiple infections were found in 21.9% (79/361) of the specimens, most prevalently in 20-29-year olds while the highest prevalence of HPV infection was found in the 30-39-year age group. The most prevalent genotype was HPV 16 at 28.2% (138/489) followed by HPV 52 at 9.6% (47/489), with the other types occurring at less than 9.0%. The electrochemical DNA chip results were compared with INNO-LiPA and sequencing (E1 and L1 regions) based on random selection of 273 specimens. The results obtained by the three methods were in agreement except for three cases. Direct sequencing detected only one predominant genotype including low risk HPV genotypes. INNO-LiPA identified multiple infections with various specific genotypes including some unclassified-risk genotypes. The electrochemical DNA chip was highly accurate, suitable for detection of single and multiple infections, allowed rapid detection, was less time-consuming and was easier to perform when compared with the other methods. It is concluded that for clinical and epidemiological studies, all genotyping methods are perfectly suitable and provide comparable results.

• Proceedings of the Zoological Society Korea Conference
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• 1995.10b
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• pp.322-322
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• 1995

• Proceedings of the Zoological Society Korea Conference
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• 1995.10b
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• pp.323.1-323
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• 1995

• Korean Journal of Agricultural Science
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• v.41 no.3
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• pp.245-249
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• 2014

Among dozens of DNA polymerases cloned from thermophilic bacteria, Taq DNA polymerase from Thermus aquaticus has been most frequently used in polymerase chain reaction (PCR) that is being applied to gene cloning, DNA sequencing, gene expression analysis, and detection of infectious and genetic diseases. Since native Taq DNA polymerase is expressed at low level in T. aquaticus, recombinant Escherichia coli system was used to produce Taq DNA polymerase in a large amount. Taq DNA polymerase was expressed as a soluble form under the control of tac promoter in E. coli, and purified by heat treatment and ion exchange chromatographies. The purified Taq DNA polymerase was nearly homogeneous and exhibited a similar DNA amplification activity with a commercial Taq DNA polymerase.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.456-459
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• 1992

The genes of Halohacteria. gvpD and gvpE. take part in formation of gas vesicle. These mRNA cause a lot of experimental prohlems due to its eharacteristic instahility in the analysis of transcripts. This study allowed easy cloning and sequencing of RNA hy substituting a stable complementary DNA for the mRNA of the genes for an analysis. The weak 111 RNA was reverse transcribed to DNA using reverse transcriptase. and was amplified using PCR method. The transcripts confirmed in this ~,tudy have not heen round in the northern hybridization covering almost all ranges of ORF of the gene. gvpD.

• Journal of Life Science
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• v.8 no.5
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• pp.571-575
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• 1998

The nerve system specific protein of Drosophila melanogaster was produced by using heads of flies as the antigen. The monoclonal antibody 6H6 recognized the disabled molecules that a kind of tyrosine kinase substrate by expres-sion cDNA library screening method. At the same time, the antibody also specifically recognized C-terminal region of disabled protein from 7427 to 8761bp by DNA sequencing. In early embryos, the localization of antigen appeared in the central nerve system. In adult flies, the antigen showed specific localization on the axon of optic nerve, cerebral nerve and thoracic nerve, and they also expressed on the muscular nerve. The molecules of disabled are expected to carry an important function in developing central nerve system. In adult flies, it is suggested that the disabled molecules have a role for muscular nerve as well as neural axon.

• Proceedings of the Microbiological Society of Korea Conference
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• 2002.10a
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• pp.15-21
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• 2002

Terminal-restriction fragment length polymorphism (T-RFLP) analysis has been optimized by using in vitro model community composed of genomic DNAs of known bacterial strains and has been applied to assess the bacterial community structure in marine sediments. The specific fluorescence-labeled terminal restriction fragments (T-RFs) between 39 and 839 base long specifying each strain were precisely measured for known bacterial strains. The addition of a co-solvent (dimethylsulfoxide or glycerol) into PCR reactions has reduced differential PCR amplification. Comparative bacterial community structure was investigated for pristine and polluted sediments. A complex T-RFLP pattern showing complex bacterial community structure was obtained in the pristine sediment, whereas simple T-RFLP pattern (low bacterial diversity) was shown in polluted sediments where caged aquaculture has been conducted for several years. The results of T-RFLP analysis were compared with that of cloning and sequencing 16S rDNA clones from the same sediments. Sequence analysis of 16S rDNA clones (72) of the pristine sediment revealed a diverse collection of lineages, largely of the class Proteobacteria ($6\%$ alpha subdivision, $46\%$ gamma subdivision, $13\%$ delta subdivision, and $3\%$ epsilon subdivision), Nitrospina $(8\%)$, high G+C gram positive $(8\%)$, Verrucomicrobia $(7\%)$, and Planctomycetes $(6\%)$. In the contaminated sediments, 17 $(59\%)$ of the 16S rDNA clones (29) were related to Campylobacter and symbiont of Rimicaris exoculata belonging to epsilon subdivision of Proteobacteria. The results obtained indicated that T-RFLP analysis is a rapid and precise technique for comparative bacterial community analysis.

• Korean Journal Plant Pathology
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• v.11 no.1
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• pp.73-79
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• 1995

Complementary DNAs (cDNAs) to the coat protein gene of an ordinary Korean strain of potato virus Y (PVY-OK) isolated from potato (cv. Superior) were synthesized and cloned into a plasmid pUC119 and sequenced. The RNA of the virus propagated in tobacco (Nicotinaa sylvestris) was extracted by the method of phenol extraction. The first strand of cDNAs to the coat protein penomic RNA of the virus was made by Moloney murine leukemia virus reverse transcriptase. The cDNA were synthesized and amplified by the method of polymerase chain reaction (PCR) using a pair of oligonucleotide primers. PVYCP3P and PVYCP3M. The size of cDNAs inserted in pUC119 plasmid was estimated as about 840 bp upon agarose gel electrophoresis. Double stranded cDNAs were transformed into the competent cell of E. coli JM109. Sequence analysis of cDNAs was conducted by the dideoxynucleotide chain termination method. Homology of cDNAs of the PVY-OK coat protein genomic RNA with those of PVY-O (Japan), PVY-T (Japan), PVY-TH (Japan), PVYN (The Netherlands),and PVYY (France) was represented as 97.3%, 88.9%, 89.3%, 89.6% and 98.5%, respectively. Homology at the amino acid level turned out to the be 97.4%, 92.5%, 92.9%, 92.9% and 98.5%, respectively.

• Genomics & Informatics
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• v.11 no.4
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• pp.272-276
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• 2013

Sequence analysis of the 16S rRNA gene has been widely used for the classification of microorganisms. However, we have been unable to clearly identify five Flavobacterium species isolated from a freshwater by using the gene as a single marker, because the evolutionary history is incomplete and the pace of DNA substitutions is relatively rapid in the bacteria. In this study, we tried to classify Flavobacterium species through multilocus sequence analysis (MLSA), which is a practical and reliable technique for the identification or classification of bacteria. The five Flavobacterium species isolated from freshwater and 37 other strains were classified based on six housekeeping genes: gyrB, dnaK, tuf, murG, atpA, and glyA. The genes were amplified by PCR and subjected to DNA sequencing. Based on the combined DNA sequence (4,412 bp) of the six housekeeping genes, we analyzed the phylogenetic relationship among the Flavobacterium species. The results indicated that MLSA, based on the six housekeeping genes, is a trustworthy method for the identification of closely related Flavobacterium species.