• International Journal of Industrial Entomology and Biomaterials
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• v.27 no.2
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• pp.271-276
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• 2013

The Asian honeybee, Apis cerana, is a native honeybee species in Korea which is important in agriculture for pollination and honey production. For better understanding of the physiology of A. cerana, high-throughput Illumina transcriptome sequencing was performed to analyze the gene expression profiles of queen, worker, and larva. A total of 219,799,682 clean reads corresponding to 22.2 Gb of nucleotide sequences was obtained from the whole body total RNA samples. The Apis mellifera reference mRNA sequence database was used to measure the gene expression level with Bowtie2 and eXpress software, and the Illumina short reads were then mapped to 11,459 out of 11,736 A. mellifera reference genes. Total of 9,221 genes with FPKM value greater than 5 of each sample group were subjected to eggNOG with BLASTX for gene ontology analysis. The differential gene expression between queen and worker, and worker and larva were analyzed to screen the overexpressed genes in each sample group. In the queen and worker sample group, total of 1,766 genes were differentially expressed with 887 and 879 genes overexpressed over two folds in queen and worker, respectively. In the worker and larva sample group, total of 1,410 genes were differentially expressed with 1,009 and 401 genes overexpressed over two folds in worker and larva, respectively.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.17
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• pp.7935-7941
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• 2015

The aim of this study was to implement massive parallel sequencing (MPS) technology in clinical genetics testing. We developed and tested an amplicon-based method for resequencing the BRCA1 and BRCA2 genes on an Illumina MiSeq to identify disease-causing mutations in patients with hereditary breast or ovarian cancer (HBOC). The coding regions of BRCA1 and BRCA2 were resequenced in 96 HBOC patient DNA samples obtained from different sample types: peripheral blood leukocytes, whole blood drops dried on paper, and buccal wash epithelia. A total of 16 random DNA samples were characterized using standard Sanger sequencing and applied to optimize the variant calling process and evaluate the accuracy of the MPS-method. The best bioinformatics workflow included the filtration of variants using GATK with the following cut-offs: variant frequency >14%, coverage ($>25{\times}$) and presence in both the forward and reverse reads. The MPS method had 100% sensitivity and 94.4% specificity. Similar accuracy levels were achieved for DNA obtained from the different sample types. The workflow presented herein requires low amounts of DNA samples (170 ng) and is cost-effective due to the elimination of DNA and PCR product normalization steps.

• Journal of Life Science
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• v.29 no.1
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• pp.1-8
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• 2019

In Hanwoo cattle (Korean native cattle), there is a scarcity of comparative analysis papers using highdepth sequencing and haplotype phasing, particularly a comparative analysis of the Truseq Synthetic Long-Read Haplotyping sequencing platform serviced by Illumina (TSLRH) versus the Chromium Genome Sequencing platform serviced by 10X Genomics (10XG). DNA was extracted from the sperm of a Hanwoo breeding bull (ID: TN1505D2184/27214) provided by Hanwoo research canter and used for the generation of sequence data from both the sequencing platforms. We then identified SNVs using an appropriate analysis pipeline tailored for each platform. The TSLRH and 10XG platforms generated a total of 355,208,304 and 1,632,772,004 reads, respectively, corresponding to a Q30 (%) of 89.04% and 88.60%, respectively, of which 351,992,768(99.09%) and 1,526,641,824(93.50%) were successfully mapped. For the TSLRH and 10XG platforms, the mean depth of the sequencing was 13.04X and 74.3X, the longest phase block was 1,982,706 bp and 1,480,081 bp, the N50 phase block was 57,637 bp and 114,394 bp, the total number of SNVs identified was 4,534,989 and 8,496,813, and the total phased rate was 72.29% and 87.67%, respectively. Moreover, for each chromosome, we identified unique and common SNVs using both sequencing platforms. The number of SNVs was directly proportional to the length of the chromosome. Based on our results, we recommend the use of the 10XG platform for haplotype phasing and SNV identification, as it generated a longer N50 phase block, in addition to a higher mean depth, total number of reads, total number of SNVs, and phase rate, than the TSLRH platform.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.1
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• pp.13-18
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• 2018

Objective: Meat quality including muscle color in chickens is an important trait and continuous selective pressures for fast growth and high yield have negatively impacted this trait. This study was conducted to investigate genetic variations responsible for regulating muscle color. Methods: Whole genome re-sequencing analysis using Illumina HiSeq paired end read method was performed with pooled DNA samples isolated from two broiler chicken lines divergently selected for muscle color (high muscle color [HMC] and low muscle color [LMC]) along with their random bred control line (RAN). Sequencing read data was aligned to the chicken reference genome sequence for Red Jungle Fowl (Galgal4) using reference based genome alignment with NGen program of the Lasergene software package. The potential causal single nucleotide polymorphisms (SNPs) showing non-synonymous changes in coding DNA sequence regions were chosen in each line. Bioinformatic analyses to interpret functions of genes retaining SNPs were performed using the ingenuity pathways analysis (IPA). Results: Millions of SNPs were identified and totally 2,884 SNPs (1,307 for HMC and 1,577 for LMC) showing >75% SNP rates could induce non-synonymous mutations in amino acid sequences. Of those, SNPs showing over 10 read depths yielded 15 more reliable SNPs including 1 for HMC and 14 for LMC. The IPA analyses suggested that meat color in chickens appeared to be associated with chromosomal DNA stability, the functions of ubiquitylation (UBC) and quality and quantity of various subtypes of collagens. Conclusion: In this study, various potential genetic markers showing amino acid changes were identified in differential meat color lines, that can be used for further animal selection strategy.

• Genomics & Informatics
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• v.13 no.3
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• pp.81-85
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• 2015

Molecular characterization technology in genetically modified organisms, in addition to how transgenic biotechnologies are developed now require full transparency to assess the risk to living modified and non-modified organisms. Next generation sequencing (NGS) methodology is suggested as an effective means in genome characterization and detection of transgenic insertion locations. In the present study, we applied NGS to insert transgenic loci, specifically the epidermal growth factor (EGF) in genetically modified rice cells. A total of 29.3 Gb (${\sim}72{\times}coverage$) was sequenced with a $2{\times}150bp$ paired end method by Illumina HiSeq2500, which was consecutively mapped to the rice genome and T-vector sequence. The compatible pairs of reads were successfully mapped to 10 loci on the rice chromosome and vector sequences were validated to the insertion location by polymerase chain reaction (PCR) amplification. The EGF transgenic site was confirmed only on chromosome 4 by PCR. Results of this study demonstrated the success of NGS data to characterize the rice genome. Bioinformatics analyses must be developed in association with NGS data to identify highly accurate transgenic sites.

• Journal of Life Science
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• v.30 no.3
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• pp.291-297
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• 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 Life Science
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• v.30 no.1
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• pp.64-69
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• 2020

The European honeybee (Apis mellifera L.) has multiple anti-microbial peptides, but many were unknown and demands for their characterization have increased. This study therefore focused on identifying novel anti-microbial peptides (AMPs) from A. mellifera L. To obtain high-throughput transcriptome data of the honeybee, we implemented next-generation sequencing (NGS), isolating novel AMPs from total RNA, and generated 15,314 peptide sequences, including 44 known, using Illumina HiSeq 2500 technology. The uncharacterized peptides were identified based on specific features of possible AMPs predicted in the sequencing analysis. AMP5, one such uncharacterized peptide, was expressed in the epidermis, body fat, and venom gland of the honeybee. We chemically synthesized this peptide and tested its anti-bacterial activity against Gram-negative Escherichia coli (KACC 10005) and Gram-positive Bacillus thuringiensis (KACC 10168) by anti-microbial assay. AMP5 exhibited anti-bacterial activity against E. coli (MIC50=22.04±0.66 μM) but not against B. thuringiensis. When worker bees were injected with E. coli, AMP5 was up-regulated in the body fat. This study therefore identified AMP5 in adult European honeybees and confirmed its anti-bacterial activity against Gram-negative E. coli.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.7
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• pp.957-966
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• 2017

Objective: To understand the dynamic structure, function, and influence on nutrient metabolism in hosts, it was crucial to assess the genetic potential of gut microbial community in yaks of different ages. Methods: The denaturing gradient gel electrophoresis (DGGE) profiles and Illumina-based metagenomic sequencing on colon contents of 15 semi-domestic yaks were investigated. Unweighted pairwise grouping method with mathematical averages (UPGMA) clustering and principal component analysis (PCA) were used to analyze the DGGE fingerprint. The Illumina sequences were assembled, predicted to genes and functionally annotated, and then classified by querying protein sequences of the genes against the Kyoto encyclopedia of genes and genomes (KEGG) database. Results: Metagenomic sequencing showed that more than 85% of ribosomal RNA (rRNA) gene sequences belonged to the phylum Firmicutes and Bacteroidetes, indicating that the family Ruminococcaceae (46.5%), Rikenellaceae (11.3%), Lachnospiraceae (10.0%), and Bacteroidaceae (6.3%) were dominant gut microbes. Over 50% of non-rRNA gene sequences represented the metabolic pathways of amino acids (14.4%), proteins (12.3%), sugars (11.9%), nucleotides (6.8%), lipids (1.7%), xenobiotics (1.4%), coenzymes, and vitamins (3.6%). Gene functional classification showed that most of enzyme-coding genes were related to cellulose digestion and amino acids metabolic pathways. Conclusion: Yaks' age had a substantial effect on gut microbial composition. Comparative metagenomics of gut microbiota in 0.5-, 1.5-, and 2.5-year-old yaks revealed that the abundance of the class Clostridia, Bacteroidia, and Lentisphaeria, as well as the phylum Firmicutes, Bacteroidetes, Lentisphaerae, Tenericutes, and Cyanobacteria, varied more greatly during yaks' growth, especially in young animals (0.5 and 1.5 years old). Gut microbes, including Bacteroides, Clostridium, and Lentisphaeria, make a contribution to the energy metabolism and synthesis of amino acid, which are essential to the normal growth of yaks.

• Korean Journal of Microbiology
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• v.52 no.3
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• pp.375-379
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• 2016

In this study, the structure and diversity of bacterial community were investigated in the surface and subsurface marine sediments using a NGS method (i.e. illumina sequencing technology). The bacterial community in the surface was distinct from that in the subsurface of marine sediment; with the exception of the phylum Proteobacteria, the relative abundance of Bacteroides phylum were higher in the surface than subsurface, whereas the sequences affiliated to the phyla Chloroflexi and Acidobacteria were relatively more copious in the subsurface than surface sediment. Moreover, interestingly, we observed that the phyla Nitrospinae and Nitrospirae contribute to nitrogen cycle in the marine sediment. This study may present the possibility for the presence of novel microorganisms as unexplored sources and provide basic information on the microbial community structure.

• Korean Journal of Plant Taxonomy
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• v.48 no.3
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• pp.195-200
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• 2018

Genetic assessments of rare and endangered species are among the first steps necessary to establish the proper management of natural populations. Transcriptome-derived single-sequence repeat markers were developed for the Korean endangered species Astilboides tabularis (Saxifragaceae) to assess its genetic diversity. A total of 96 candidate microsatellite loci were isolated based on transcriptome data using Illumina pair end sequencing. Of these, 26 were polymorphic, with one to five alleles per locus in 60 individuals from three populations of A. tabularis. The observed and expected heterozygosity per locus ranged from 0.000 to 0.950 and from 0.000 to 0.741, respectively. These polymorphic transcriptome-derived simple sequence repeat markers would be invaluable for future studies of population genetics and for ecological conservation of the endangered species A. tabularis.