• Journal of Life Science
• /
• v.31 no.4
• /
• pp.410-417
• /
• 2021

Next-generation sequencing (NGS) is a high-throughput technique for sequencing large numbers of DNA fragments that are prepared from a genome. This sequencing technique has been used to elucidate whole genome sequences of living organisms and to analyze complementary DNA (cDNA) or chromatin immunoprecipitated DNA (ChIPed DNA) at the genome level. After NGS, the use of proper tools is important for processing and analyzing data with reasonable parameters. However, handling large-scale sequencing data and programing for data analysis can be difficult. The Galaxy platform, a public web service system, provides many different tools for NGS data analysis, and it allows researchers to analyze their data on a web browser with no deep knowledge about bioinformatics and/or programing. In this study, we explain the procedure for preparing chromatin immunoprecipitation-sequencing (ChIP-seq) libraries and steps for analyzing ChIP-seq data using the Galaxy platform. The data analysis steps include the NGS data upload to Galaxy, quality check of the NGS data, premapping processes, read mapping, the post-mapping process, peak-calling and visualization by window view, heatmaps, average profile, and correlation analysis. Analysis of our histone H3K4me1 ChIP-seq data in K562 cells shows that it correlates with public data. Thus, NGS data analysis using the Galaxy platform can provide an easy approach to bioinformatics.

• The Korean Journal of Malacology
• /
• v.31 no.3
• /
• pp.243-247
• /
• 2015

A stand-alone BLAST server is available that provides a convenient and amenable platform for the analysis of molluscan sequence information especially the EST sequences generated by traditional sequencing methods. However, it is found that the server has limitations in the annotation of molluscan sequences generated using next-generation sequencing (NGS) platforms due to inconsistencies in molluscan sequence available at NCBI. We constructed a web-based interface for a new stand-alone BLAST, called PANM-DB (Protostome DB) for the analysis of molluscan NGS data. The PANM-DB includes the amino acid sequences from the protostome groups-Arthropoda, Nematoda, and Mollusca downloaded from GenBank with the NCBI taxonomy Browser. The sequences were translated into multi-FASTA format and stored in the database by using the formatdb program at NCBI. PANM-DB contains 6% of NCBInr database sequences (as of 24-06-2015), and for an input of 10,000 RNA-seq sequences the processing speed was 15 times faster by using PANM-DB when compared with NCBInr DB. It was also noted that PANM-DB show two times more significant hits with diverse annotation profiles as compared with Mollusks DB. Hence, the construction of PANM-DB is a significant step in the annotation of molluscan sequence information obtained from NGS platforms. The PANM-DB is freely downloadable from the web-based interface (Malacological Society of Korea, http://malacol.or/kr/blast) as compressed file system and can run on any compatible operating system.

• Journal of Gastric Cancer
• /
• v.20 no.1
• /
• pp.29-40
• /
• 2020

Purpose: Gastrointestinal stromal tumors (GISTs) frequently harbor activating gene mutations in either KIT or platelet-derived growth factor receptor A (PDGFRA) and are highly responsive to several selective tyrosine kinase inhibitors. In this study, a targeted next-generation sequencing (NGS) assay with an Oncomine Focus Assay (OFA) panel was used for the genetic characterization of molecular targets in 30 Korean patients with GIST. Materials and Methods: Using the OFA that enables rapid and simultaneous detection of hotspots, single nucleotide variants (SNVs), insertion and deletions (Indels), copy number variants (CNVs), and gene fusions across 52 genes relevant to solid tumors, targeted NGS was performed using genomic DNA extracted from formalin-fixed and paraffin-embedded samples of 30 GISTs. Results: Forty-three hotspot/other likely pathogenic variants (33 SNVs, 8 Indels, and 2 amplifications) in 16 genes were identified in 26 of the 30 GISTs. KIT variants were most frequent (44%, 19/43), followed by 6 variants in PIK3CA, 3 in PDGFRA, 2 each in JAK1 and EGFR, and 1 each in AKT1, ALK, CCND1, CTNNB1, FGFR3, FGFR4, GNA11, GNAQ, JAK3, MET, and SMO. Based on the mutation types, majority of the variants carried missense mutations (60%, 26/43), followed by 8 frameshifts, 6 nonsense, 1 stop-loss, and 2 amplifications. Conclusions: Our study confirmed the advantage of using targeted NGS with a cancer gene panel to efficiently identify mutations associated with GISTs. These findings may provide a molecular genetic basis for developing new drugs targeting these gene mutations for GIST therapy.

• Journal of the Korea Institute of Information and Communication Engineering
• /
• v.17 no.12
• /
• pp.3009-3015
• /
• 2013

Next-generation sequencing (NGS) has enabled whole genome and transcriptome single nucleotide variant (SNV) discovery in cancer and method of the most fundamental being determining an individual's genotype from multiple aligned short read sequences at a position. Bayesian algorithm estimate parameter using posterior genotype probabilities and other method, EM algorithm, estimate parameter using maximum likelihood estimate method in observed data. Here, we propose a novel genotype-calling system and compare and analyze the effect of sample size(S = 50, 100 and 500) on posterior estimate of sequencing error rate, somatic mutation status and genotype probability. The result is that estimate applying Bayesian algorithm even for 50 of small sample size approached real parameter than estimate applying EM algorithm in small sample more accurately.

• Genomics & Informatics
• /
• v.15 no.4
• /
• pp.136-141
• /
• 2017

Accurate detection of genomic alterations, especially druggable hotspot mutations in tumors, has become an essential part of precision medicine. With targeted sequencing, we can obtain deeper coverage of reads and handle data more easily with a relatively lower cost and less time than whole-exome or whole-genome sequencing. Recently, we designed a customized gene panel for targeted sequencing of major solid cancers. In this study, we aimed to validate its performance. The cancer panel targets 95 cancer-related genes. In terms of the limit of detection, more than 86% of target mutations with a mutant allele frequency (MAF) <1% can be identified, and any mutation with >3% MAF can be detected. When we applied this system for the analysis of Acrometrix Oncology Hotspot Control DNA, which contains more than 500 COSMIC mutations across 53 genes, 99% of the expected mutations were robustly detected. We also confirmed the high reproducibility of the detection of mutations in multiple independent analyses. When we explored copy number alterations (CNAs), the expected CNAs were successfully detected, and this result was confirmed by target-specific genomic quantitative polymerase chain reaction. Taken together, these results support the reliability and accuracy of our cancer panel in detecting mutations. This panel could be useful for key mutation profiling research in solid tumors and clinical translation.

• Journal of Genetic Medicine
• /
• v.7 no.2
• /
• pp.119-124
• /
• 2010

More than 7,000 rare Mendelian diseases have been reported, but less than half of all rare monogenic disorders has been discovered. In addition, the majority of mutations that are known to cause Mendelian disorders are located in protein-coding regions. Therefore, exome sequencing is an efficient strategy to selectively sequence the coding regions of the human genome to identify novel genes associated with rare genetic disorders. The "exome" represents all of the exons in the human genome, constituting about 1.5% of the human genome. Exome sequencing is carried out by targeted capture and intense parallel sequencing. After the first report of successful exome sequencing for the identification of causal genes and mutations in Freeman Sheldon syndrome, exome sequencing has become a standard approach to identify genes in rare Mendelian disorders. Exome sequencing is also used to search the causal genes and variants in complex diseases. The successful use of exome sequencing in Mendelian disorders and complex diseases will facilitate the development of personalized genomic medicine.

• Korean Journal of Soil Science and Fertilizer
• /
• v.46 no.3
• /
• pp.173-181
• /
• 2013

Livestock wastes are considered as major environmental pollutants because they contain high concentration of organic materials. In 2001, The Environmental Department reported that stock farmers were increasing as 5.1%/year, which resulted in a gradual increase in livestock wastes generation. The direct disposal of livestock wastes create several environmental problems. Thus, several countries banned the disposal of livestock wastes in environment including aquatic systems. Recently, aeration-based liquid fertilization was considered as potential way for the disposal of livestock wastes. In this study, next generation sequencing (NGS) analysis was used to understand the microbial community changes during liquid fertilization of livestock wastes. Microbial community was compared with liquid fertilizer physicochemical analysis such as $BOD_5$, $COD_{Mn}$ pH, N (Nitrogen), P (Phosphorus), K (Potassium) etc. The physicochemical parameters and bacterial community results pave the way for producing effective livestock-based fertilizer. By comparing the physical characteristics of the manure with microbial community changes, it is possible to optimize the conditions for producing effective fertilizer.

• Korean Journal of Veterinary Service
• /
• v.45 no.1
• /
• pp.31-38
• /
• 2022

Swine influenza (SI) is an important respiratory disease in pigs and epidemic worldwide, which is caused by influenza A virus (IAV) belonging to the family of Orthomyxoviridae. As seen again in the 2009 swine-origin influenza A H1N1 pandemic, pigs are known to be susceptible to swine, avian, and human IAVs, and can serve as a 'mixing vessel' for the generation of novel IAV variants. To this end, the emergence of swine influenza viruses must be kept under close surveillance. Herein, we report the isolation and phylogenetic study of a swine IAV, A/swine/Korea/21810/2021 (sw21810, H3N2 subtype). BLASTN sequence analysis of 8 gene segments of the isolated virus revealed a high degree of nucleotide similarity (94.76 to 100%) to porcine strains circulating in Korea and the United States. Out of 8 genome segments, the HA gene was closely related to that of isolates from cluster I. Additionally, the NA gene of the isolate belonged to a Korean Swine H1N1 origin, and the PB2, PB1, NP and NS genes of the isolate were grouped into that of the Triple reassortant swine H3N2 origin virus. The PA and M genes of the isolate belonged to 2009 Pandemic H1N1 lineage. Human infection with mutants was most common through contact with infected pigs. Our results suggest the need for periodic close monitoring of this novel swine H3N2 influenza virus from a public health perspective.

• Journal of Life Science
• /
• v.30 no.3
• /
• pp.291-297
• /
• 2020

This study was conducted to develop microsatellite markers in Seriola quinqueradiata using next-generation sequencing. A total of 28,873,374 reads were generated on an Illumina Hiseq2500 system, yielding 7,247,216,874 bp sequences. The de novo assembly resulted in 466,359 contigs. A total of 132 contigs (0.43%), including 60 microsatellite loci, were derived from 30,729 contigs longer than 518 bp. A total of 60 primer sets were designed from the 132 microsatellite loci. A total of 15 polymorphic nuclear microsatellite loci were chosen to evaluate population genetic parameters in the parents and offspring. The mean number of effective alleles was 18.5, ranging from 11 to 30. The observed heterozygosity (H_O) and expected heterozygosity (H_E) ranged between 0.431 and 0.972 with an average of 0.812 and from 0.782 to 0.949 with an average of 0.896, respectively. No significant linkage disequilibrium was observed after Bonferroni revision in any loci. The results show that the 15 polymorphic nuclear microsatellite markers can be used to study the population and conservation genetics of S. quinqueradiata in Korea. To ensure the success of artificial seedling production technology, genetic variations between the parent and offspring populations should be monitored, and inbreeding should be controlled.

• Journal of Korean Society of Forest Science
• /
• v.105 no.2
• /
• pp.186-192
• /
• 2016

This study was conducted to develop microsatellite markers in Quercus variabilis using next generation sequencing. A total of 305,771 reads (384 bp on average) were generated on a Roche GS-FLX system, yielding 117 Mbp of sequences. The de novo assembly resulted in 7,346 contigs. A total of 606 contigs (20.75%) including 911 microsatellite loci were derived from the 2,921 contigs longer than 500 bp. A total of 180 primer sets were designed from the 911 microsatellite loci and screened in eight Q. variabilis individual trees sampled from a natural stand to obtain polymorphic loci. As a result, a total of thirteen polymorphic microsatellite loci were selected and used for estimating population genetic parameters in the 54 individual trees. The mean number of effective alleles was 4.996 ranging from 2.439 to 7.515. The observed heterozygosity and the expected heterozygosity ranged between 0.731 and 1.000 with an average of 0.873 and from 0.590 to 0.867 with an average of 0.766, respectively. Null alleles were not detected in all loci. No significant linkage disequilibrium was detected after Bonferroni correction in all loci. In the near future, these novel polymorphic microsatellite markers will be used to study population and conservation genetics of Q. variabilis of Korea in more detail.