• Journal of Microbiology and Biotechnology
• /
• v.25 no.9
• /
• pp.1410-1416
• /
• 2015

Wolbachia is an obligate symbiotic bacteria that is ubiquitous in arthropods, with 25-70% of insect species estimated to be infected. Wolbachia species can interact with their insect hosts in a mutualistic or parasitic manner. Sequence types (ST) of Wolbachia are determined by multilocus sequence typing (MLST) of housekeeping genes. However, there are some limitations to MLST with respect to the generation of clone libraries and the Sanger sequencing method when a host is infected with multiple STs of Wolbachia. To assess the feasibility of massive parallel sequencing, also known as next-generation sequencing, we used pyrosequencing for sequence typing of Wolbachia in butterflies. We collected three species of butterflies (Eurema hecabe, Eurema laeta, and Tongeia fischeri) common to Korea and screened them for Wolbachia STs. We found that T. fischeri was infected with a single ST of Wolbachia, ST41. In contrast, E. hecabe and E. laeta were each infected with two STs of Wolbachia, ST41 and ST40. Our results clearly demonstrate that pyrosequencing-based MLST has a higher sensitivity than cloning and Sanger sequencing methods for the detection of minor alleles. Considering the high prevalence of infection with multiple Wolbachia STs, next-generation sequencing with improved analysis would assist with scaling up approaches to Wolbachia MLST.

• Genomics & Informatics
• /
• v.16 no.2
• /
• pp.22-29
• /
• 2018

Incorporation of unique barcodes into fission yeast gene deletion collections has enabled the identification of gene functions by growth fitness analysis. For fine tuning, it is important to examine barcode sequences, because mutations arise during strain construction. Out of 8,708 barcodes (4,354 strains) covering 88.5% of all 4,919 open reading frames, 7,734 barcodes (88.8%) were validated as high-fidelity to be inserted at the correct positions by Sanger sequencing. Sequence examination of the 7,734 high-fidelity barcodes revealed that 1,039 barcodes (13.4%) deviated from the original design. In total, 1,284 mutations (mutation rate of 16.6%) exist within the 1,039 mutated barcodes, which is comparable to budding yeast (18%). When the type of mutation was considered, substitutions accounted for 845 mutations (10.9%), deletions accounted for 319 mutations (4.1%), and insertions accounted for 121 mutations (1.6%). Peculiarly, the frequency of substitutions (67.6%) was unexpectedly higher than in budding yeast (~28%) and well above the predicted error of Sanger sequencing (~2%), which might have arisen during the solid-phase oligonucleotide synthesis and PCR amplification of the barcodes during strain construction. When the mutation rate was analyzed by position within 20-mer barcodes using the 1,284 mutations from the 7,734 sequenced barcodes, there was no significant difference between up-tags and down-tags at a given position. The mutation frequency at a given position was similar at most positions, ranging from 0.4% (32/7,734) to 1.1% (82/7,734), except at position 1, which was highest (3.1%), as in budding yeast. Together, well-defined barcode sequences, combined with the next-generation sequencing platform, promise to make the fission yeast gene deletion library a powerful tool for understanding gene function.

• Genomics & Informatics
• /
• v.19 no.4
• /
• pp.41.1-41.10
• /
• 2021

In addition to mutations and copy number alterations, gene fusions are commonly identified in cancers. In thyroid cancer, fusions of important cancer-related genes have been commonly reported; however, extant panels do not cover all clinically important gene fusions. In this study, we aimed to develop a custom RNA-based sequencing panel to identify the key fusions in thyroid cancer. Our ThyChase panel was designed to detect 87 types of gene fusion. As quality control of RNA sequencing, five housekeeping genes were included in this panel. When we applied this panel for the analysis of fusions containing reference RNA (HD796), three expected fusions (EML4-ALK, CCDC6-RET, and TPM3-NTRK1) were successfully identified. We confirmed the fusion breakpoint sequences of the three fusions from HD796 by Sanger sequencing. Regarding the limit of detection, this panel could detect the target fusions from a tumor sample containing a 1% fusion-positive tumor cellular fraction. Taken together, our ThyChase panel would be useful to identify gene fusions in the clinical field.

• Annals of Laboratory Medicine
• /
• v.38 no.6
• /
• pp.545-554
• /
• 2018

Background: The increasing morbidity and mortality rates associated with Acinetobacter baumannii are due to the emergence of drug resistance and the limited treatment options. We compared characteristics of colistin-resistant Acinetobacter baumannii (CR-AB) clinical isolates recovered from patients with and without prior colistin treatment. We assessed whether prior colistin treatment affects the resistance mechanism of CR-AB isolates, mortality rates, and clinical characteristics. Additionally, a proper method for identifying CR-AB was determined. Methods: We collected 36 non-duplicate CR-AB clinical isolates resistant to colistin. Antimicrobial susceptibility testing, Sanger sequencing analysis, molecular typing, lipid A structure analysis, and in vitro synergy testing were performed. Eleven colistin-susceptible AB isolates were used as controls. Results: Despite no differences in clinical characteristics between patients with and without prior colistin treatment, resistance-causing genetic mutations were more frequent in isolates from colistin-treated patients. Distinct mutations were overlooked via the Sanger sequencing method, perhaps because of a masking effect by the colistin-susceptible AB subpopulation of CR-AB isolates lacking genetic mutations. However, modified lipid A analysis revealed colistin resistance peaks, despite the population heterogeneity, and peak levels were significantly different between the groups. Conclusions: Although prior colistin use did not induce clinical or susceptibility differences, we demonstrated that identification of CR-AB by sequencing is insufficient. We propose that population heterogeneity has a masking effect, especially in colistin non-treated patients; therefore, accurate testing methods reflecting physiological alterations of the bacteria, such as phosphoethanolamine-modified lipid A identification by matrix-assisted laser desorption ionization-time of flight, should be employed.

• Korean Journal of Soil Science and Fertilizer
• /
• v.45 no.6
• /
• pp.1073-1085
• /
• 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.

• Proceedings of the Ginseng society Conference
• /
• 1990.06a
• /
• pp.65-67
• /
• 1990

The sequence of ginseng peptide gene was designed and synthesized by the solid phase plasmid pUC19. Escherichia coli JM101 cells were transformed with above hybrid plasmids. Ampicillin resistant transformants were screened and identified by in situ colony hybridization and Southern blot techinques. Finally the gene sequencing was done by the Sanger dideoxy method using primer extension.

• Journal of Ginseng Research
• /
• v.14 no.2
• /
• pp.207-209
• /
• 1990

The sequence of ginseng peptide gene was designed and synthesized by the solid phase phosphoramidiate method. Synthetic segments were isolated, pllrified and joined to the plasmid pUC19. E.icherichiu coli JM101 cells were transformed with above hybrid plasmids. AmpiciIBin resistant transformants were screened and identified by in situ colony hybridization and Southern blot techniques. Finally the gene sequencing was done by the Sanger dideoxy method using primer extension.

• Clinical and Experimental Pediatrics
• /
• v.58 no.9
• /
• pp.317-324
• /
• 2015

Kabuki syndrome (KS) is a rare syndrome characterized by multiple congenital anomalies and mental retardation. Other characteristics include a peculiar facial gestalt, short stature, skeletal and visceral abnormalities, cardiac anomalies, and immunological defects. Whole exome sequencing has uncovered the genetic basis of KS. Prior to 2013, there was no molecular genetic information about KS in Korean patients. More recently, direct Sanger sequencing and exome sequencing revealed KMT2D variants in 11 Korean patients and a KDM6A variant in one Korean patient. The high detection rate of KMT2D and KDM6A mutations (92.3%) is expected owing to the strict criteria used to establish a clinical diagnosis. Increased awareness and understanding of KS among clinicians is important for diagnosis and management of KS and for primary care of KS patients. Because mutation detection rates rely on the accuracy of the clinical diagnosis and the inclusion or exclusion of atypical cases, recognition of KS will facilitate the identification of novel mutations. A brief review of KS is provided, highlighting the clinical and genetic characteristics of patients with KS.

• Journal of Genetic Medicine
• /
• v.15 no.2
• /
• pp.110-114
• /
• 2018

Ataxia-telangiectasia (AT; OMIM 208900) is a rare autosomal recessive inherited progressive neurodegenerative disorder, with onset in early childhood. AT is caused by homozygous or compound heterozygous mutations in ATM (OMIM 607585) on chromosome 11q22. The average prevalence of the disease is estimated at 1 of 100,000 children worldwide. The prevalence of AT in the Republic of Korea is suggested to be extremely low, with only a few cases genetically confirmed thus far. Herein, we report a 5-year-old Korean boy with clinical features such as progressive gait and truncal ataxia, both ankle spasticity, dysarthria, and mild intellectual disability. The patient was identified as a compound heterozygote with two novel genetic variants: a paternally derived c.5288_5289insGA p.(Tyr1763*) nonsense variant and a maternally derived c.8363A>C p.(His2788Pro) missense variant, as revealed by next-generation sequencing and confirmed by Sanger sequencing. Based on claims data from the Health Insurance Review and Assessment Service Republic of Korea, we calculated the prevalence of AT in the Republic of Korea to be about 0.9 per million individuals, which is similar to the worldwide average. Therefore, we suggest that multi-gene panel sequencing including ATM should be considered early diagnosis.

• Journal of Genetic Medicine
• /
• v.16 no.1
• /
• pp.27-30
• /
• 2019

Smith-Kingsmore syndrome (SKS; OMIM 616638), also known as macrocephaly-intellectual disability-neurodevelopmental disorder-small thorax syndrome (MINDS; ORPHA 457485), is a rare autosomal dominant disorder, the prevalence of which is not known. It is caused by a heterozygous germline mutation in MTOR (OMIM 601231). Ten different MTOR germline mutations in 27 individuals have been reported in the medical literature to date. These were all gain-of-function missense variants, and about half of the 27 individuals had c.5395G>A p.(Glu1799Lys) in MTOR. Here, I report for the first time a Korean patient with the heterozygous germline mutation c.5395G>A p.(Glu1799Lys) in MTOR. It was found to be a de novo mutation, which was identified by whole-exome sequencing and confirmed by Sanger sequencing. The patient showed typical clinical features of SKS, including macrocephaly/megalencephaly; moderate intellectual disability; seizures; behavioral problems; and facial dysmorphic features of curly hair, frontal bossing, midface hypoplasia, and hypertelorism.