• 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.

• Biomedical Science Letters
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• v.24 no.4
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• pp.292-304
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• 2018

Microbes is becoming increasingly forensic possibility as a consequence of advances in massive parallel sequencing (MPS) and bioinformatics. Human DNA typing is the best identifier, but it is not always possible to extract a full DNA profile namely its degradation and low copy number, and it may have limitations for identical twins. To overcome these unsatisfactory limitations, forensic potential for bacteria found in evidence could be used to differentiate individuals. Prokaryotic cells have a cell wall that better protects the bacterial nucleoid compared to the cell membrane of eukaryotic cells. Humans have an extremely diverse microbiome that may prove useful in determining human identity and may even be possible to link the microbes to the person responsible for them. Microbial composition within the human microbiome varies across individuals. Therefore, MPS of human microbiome could be used to identify biological samples from the different individuals, specifically for twins and other cases where standard DNA typing doses not provide satisfactory results due to degradation of human DNA. Microbial forensics is a new discipline combining forensic science and microbiology, which can not to replace current STR analysis methods used for human identification but to be complementary. Among the fields of microbial forensics, this paper will briefly describe information on the current status of microbiome research such as metagenomic code, salivary microbiome, pubic hair microbiome, microbes as indicators of body fluids, soils microbes as forensic indicator, and review microbial forensics as the feasibility of microbiome-based human identification.

• Journal of Genetic Medicine
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• v.12 no.1
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• pp.12-18
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• 2015

Malformations of cortical development (MCD) cover a broad spectrum of developmental disorders which cause the various clinical manifestations including epilepsy, developmental delay, and intellectual disability. MCD have been clinically classified based on the disruption of developmental processes such as proliferation, migration, and organization. Molecular genetic studies of MCD have improved our understanding of these disorders at a molecular level beyond the clinical classification. These recent advances are resulted from the development of massive parallel sequencing technology, also known as next-generation sequencing (NGS), which has allowed researchers to uncover novel molecular genetic pathways associated with inherited or de novo mutations. Although an increasing number of disease-related genes or genetic variations have been identified, genotype-phenotype correlation is hampered when the biological or pathological functions of identified genetic variations are not fully understood. To elucidate the causality of genetic variations, in vivo disease models that reflect these variations are required. In the current review, we review the use of NGS technology to identify genes involved in MCD, and discuss how the functions of these identified genes can be validated through in vivo disease modeling.

• Microbiology and Biotechnology Letters
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• v.48 no.4
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• pp.574-581
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• 2020

Next generation sequencing is commonly used to characterize the microbiome structure. MiSeq is commonly used to analyze the microbiome due to its relatively long read length. However, recently, Illumina introduced the 250x2 chip for HiSeq 2500. The purpose of this study was to compare the performance of MiSeq and HiSeq in the context of oral microbiome samples. The MiSeq Reagent Kit V3 and the HiSeq Rapid SBS Kit V2 were used for MiSeq and HiSeq 2500 analyses, respectively. Total read count, read quality score, relative bacterial abundance, community diversity, and relative abundance correlation were analyzed. HiSeq produced significantly more read sequences and assigned taxa compared to MiSeq. Conversely, community diversity was similar in the context of MiSeq and HiSeq. However, depending on the relative abundance, the correlation between the two platforms differed. The correlation between HiSeq and MiSeq sequencing data for highly abundant taxa (> 2%), low abundant taxa (2-0.2%), and rare taxa (0.2% >) was 0.994, 0.860, and 0.416, respectively. Therefore, HiSeq 2500 may also be compatible for microbiome studies. Importantly, the HiSeq platform may allow a high-resolution massive parallel sequencing for the detection of rare taxa.

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

• Journal of Marine Life Science
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• v.6 no.1
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• pp.9-22
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• 2021

We determined the composition of water and sediment bacterial assemblages from the East Sea using 16S rRNA gene sequencing. Total bacterial reads were greater in surface waters (<100 m) than in deep seawaters (>500 m) and sediments. However, total OTUs, bacterial diversity, and evenness were greater in deep seawaters than in surface waters with those in the sediment comparable to the deep sea waters. Proteobacteria was the most dominant bacterial phylum comprising 67.3% of the total sequence reads followed by Bacteriodetes (15.8%). Planctomycetes, Verrucomicrobia, and Actinobacteria followed all together consisting of only 8.1% of the total sequence. Candidatus Pelagibacter ubique considered oligotrophic bacteria, and Planctomycetes copiotrophic bacteria showed an opposite distribution in the surface waters, suggesting a potentially direct competition for available resources by these bacteria with different traits. The bacterial community in the warm surface waters were well separated from the other deep cold seawater and sediment samples. The bacteria exclusively associated with deep sea waters was Actinobacteriacea, known to be prevalent in the deep photic zone. The bacterial group Chromatiales and Lutibacter were those exclusively associated with the sediment samples. The overall bacterial community showed similarities in the horizontal rather than vertical direction in the East Sea.

• Analytical Science and Technology
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• v.31 no.2
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• pp.65-70
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• 2018

This study compared two methods for preparing ancient DNA (aDNA) for the construction of successful shotgun libraries that may be applied to massive parallel sequencing. For the comparative analysis, the DNA of prehistoric rib samples from Hungary was extracted using either a manually prepared silica suspension or the Amicon Ultracel-15 10K ultracentrifugal device (Millipore). After the extraction of the same amount of bone powder (about 150 mg) from three samples by each method, the amount of extracted double-stranded DNA and the subsequent degree of construction of the shotgun library were analyzed. The Amicon device method was rapid and easier to perform and resulted in an approximately 11-fold higher DNA recovery than that obtained using the silica suspension. The shotgun library constructed using DNA templates prepared by the Amicon device was more successful than that constructed from templates isolated using the silica suspension. The comparative study of these two aDNA extraction methods showed that the Amicon device has the advantages of saving time, process simplicity, and high efficiency.

• Journal of Microbiology and Biotechnology
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• v.22 no.2
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• pp.159-165
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• 2012

The rhizobacterial composition varies according to the soil properties. To test if the effect of herbicides on the rhizobacterial communities of genetically modified NK603 glyphosate-tolerant maize varies according to different soil locations, a comparison was made between the effects of glyphosate (Roundup Plus), a post-emergence applied herbicide, and a pre-emergence applied herbicide (GTZ) versus untreated soil. The potential effect was monitored by direct amplification, cloning, and sequencing of the soil DNA encoding 16S rRNA, and high-throughput DNA pyrosequencing of the bacterial DNA coding for the 16S rRNA hypervariable V6 region. The results obtained using three different methods to analyze the herbicide effect on the rhizobacterial communities of genetically modified NK603 maize were comparable to those previously obtained when glyphosate-tolerant maize was grown in soil with different characteristics. Both herbicides decreased the bacterial diversity in the rhizosphere, with Actinobacteria being the taxonomic group most affected. The results suggest that both herbicides affected the structure of the maize rhizobacterial community, but glyphosate was environmentally less aggressive.

• Journal of Microbiology and Biotechnology
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• v.33 no.11
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• pp.1495-1505
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• 2023

The western honeybee Apis mellifera L., a vital crop pollinator and producer of honey and royal jelly, faces numerous threats including diseases, chemicals, and mite infestations, causing widespread concern. While extensive research has explored the link between gut microbiota and their hosts. However, the impact of Varroa destructor infestation remains understudied. In this study, we employed massive parallel amplicon sequencing assays to examine the diversity and structure of gut microbial communities in adult bee groups, comparing healthy (NG) and Varroa-infested (VG) samples. Additionally, we analyzed Varroa-infested hives to assess the whole body of larvae. Our results indicated a notable prevalence of the genus Bombella in larvae and the genera Gillamella, unidentified Lactobacillaceae, and Snodgrassella in adult bees. However, no statistically significant difference was observed between NG and VG. Furthermore, our PICRUSt analysis demonstrated distinct KEGG classification patterns between larval and adult bee groups, with larvae displaying a higher abundance of genes involved in cofactor and vitamin production. Notably, despite the complex nature of the honeybee bacterial community, methanogens were found to be present in low abundance in the honeybee microbiota.

• BMB Reports
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• v.43 no.9
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• pp.635-641
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• 2010

Piwi proteins and Piwi-interacting RNAs (piRNAs) have been implicated in transposon control in germline from Drosophila to mammals. To examine the profile of small RNA expression in human cancer cells and explore difference in small RNA transcriptome, small RNA libraries prepared from wildtype, HILI overexpressed and HILI knockdowned Hela cells were sequenced using Solexa technology. piRNAs and other repeat-associated small RNAs were observed in Hela cells. By using in situ hybridization, piR-49322 was localized in the nucleolus and around the periphery of nuclear membrane in Hela cells. Following the overexpression of HILI, the retrotransposon elements LINE1 was significantly repressed, while LINE1-associated small RNAs decreased in abundance. The present study demonstrated that HILI along with piRNAs plays a role in LINE1 suppression in Hela cancer cell line.