• Title, Summary, Keyword: RNA sequencing

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Type-specific Amplification of 5S rRNA from Panax ginseng Cultivars Using Touchdown (TD) PCR and Direct Sequencing

  • Sun, Hun;Wang, Hong-Tao;Kwon, Woo-Saeng;Kim, Yeon-Ju;Yang, Deok-Chun
    • Journal of Ginseng Research
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    • v.33 no.1
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    • pp.55-58
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    • 2009
  • Generally, the direct sequencing through PCR is faster, easier, cheaper, and more practical than clone sequencing. Frequently, standard PCR amplification is usually interpreted by mispriming internal or external regions of the target template. Normally, DNA fragments were eluted from the gel using Gel extraction kit and subjected to direct sequencing or cloning sequencing. Cloning sequencing has often troublesome and needs more time to analyze for many samples. Since touchdown (TD) PCR can generate sufficient and highly specific amplification, it reduces unwanted amplicon generation. Accordingly, TD PCR is a good method for direct sequencing due to amplifying wanted fragment. In plants the 5S-rRNA gene is separated by simple spacers. The 5S-rRNA gene sequence is very well-conserved between plant species while the spacer is species-specific. Therefore, the sequence has been used for phylogenetic studies and species identification. But frequent occurrences of spurious bands caused by complex genomes are encountered in the product spectrum of standard PCR amplification. In conclusion, the TD PCR method can be applied easily to amplify main 5S-rRNA and direct sequencing of panax ginseng cultivars.

Analysis of Whole Transcriptome Sequencing Data: Workflow and Software

  • Yang, In Seok;Kim, Sangwoo
    • Genomics & Informatics
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    • v.13 no.4
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    • pp.119-125
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    • 2015
  • RNA is a polymeric molecule implicated in various biological processes, such as the coding, decoding, regulation, and expression of genes. Numerous studies have examined RNA features using whole transcriptome sequencing (RNA-seq) approaches. RNA-seq is a powerful technique for characterizing and quantifying the transcriptome and accelerates the development of bioinformatics software. In this review, we introduce routine RNA-seq workflow together with related software, focusing particularly on transcriptome reconstruction and expression quantification.

COEX-Seq: Convert a Variety of Measurements of Gene Expression in RNA-Seq

  • Kim, Sang Cheol;Yu, Donghyeon;Cho, Seong Beom
    • Genomics & Informatics
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    • v.16 no.4
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    • pp.36.1-36.3
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    • 2018
  • Next generation sequencing (NGS), a high-throughput DNA sequencing technology, is widely used for molecular biological studies. In NGS, RNA-sequencing (RNA-Seq), which is a short-read massively parallel sequencing, is a major quantitative transcriptome tool for different transcriptome studies. To utilize the RNA-Seq data, various quantification and analysis methods have been developed to solve specific research goals, including identification of differentially expressed genes and detection of novel transcripts. Because of the accumulation of RNA-Seq data in the public databases, there is a demand for integrative analysis. However, the available RNA-Seq data are stored in different formats such as read count, transcripts per million, and fragments per kilobase million. This hinders the integrative analysis of the RNA-Seq data. To solve this problem, we have developed a web-based application using Shiny, COEX-seq (Convert a Variety of Measurements of Gene Expression in RNA-Seq) that easily converts data in a variety of measurement formats of gene expression used in most bioinformatic tools for RNA-Seq. It provides a workflow that includes loading data set, selecting measurement formats of gene expression, and identifying gene names. COEX-seq is freely available for academic purposes and can be run on Windows, Mac OS, and Linux operating systems. Source code, sample data sets, and supplementary documentation are available as well.

Identification of Alternative Splicing and Fusion Transcripts in Non-Small Cell Lung Cancer by RNA Sequencing

  • Hong, Yoonki;Kim, Woo Jin;Bang, Chi Young;Lee, Jae Cheol;Oh, Yeon-Mok
    • Tuberculosis and Respiratory Diseases
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    • v.79 no.2
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    • pp.85-90
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    • 2016
  • Background: Lung cancer is the most common cause of cancer related death. Alterations in gene sequence, structure, and expression have an important role in the pathogenesis of lung cancer. Fusion genes and alternative splicing of cancer-related genes have the potential to be oncogenic. In the current study, we performed RNA-sequencing (RNA-seq) to investigate potential fusion genes and alternative splicing in non-small cell lung cancer. Methods: RNA was isolated from lung tissues obtained from 86 subjects with lung cancer. The RNA samples from lung cancer and normal tissues were processed with RNA-seq using the HiSeq 2000 system. Fusion genes were evaluated using Defuse and ChimeraScan. Candidate fusion transcripts were validated by Sanger sequencing. Alternative splicing was analyzed using multivariate analysis of transcript sequencing and validated using quantitative real time polymerase chain reaction. Results: RNA-seq data identified oncogenic fusion genes EML4-ALK and SLC34A2-ROS1 in three of 86 normal-cancer paired samples. Nine distinct fusion transcripts were selected using DeFuse and ChimeraScan; of which, four fusion transcripts were validated by Sanger sequencing. In 33 squamous cell carcinoma, 29 tumor specific skipped exon events and six mutually exclusive exon events were identified. ITGB4 and PYCR1 were top genes that showed significant tumor specific splice variants. Conclusion: In conclusion, RNA-seq data identified novel potential fusion transcripts and splice variants. Further evaluation of their functional significance in the pathogenesis of lung cancer is required.

Dissecting Cellular Heterogeneity Using Single-Cell RNA Sequencing

  • Choi, Yoon Ha;Kim, Jong Kyoung
    • Molecules and Cells
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    • v.42 no.3
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    • pp.189-199
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    • 2019
  • Cell-to-cell variability in gene expression exists even in a homogeneous population of cells. Dissecting such cellular heterogeneity within a biological system is a prerequisite for understanding how a biological system is developed, homeostatically regulated, and responds to external perturbations. Single-cell RNA sequencing (scRNA-seq) allows the quantitative and unbiased characterization of cellular heterogeneity by providing genome-wide molecular profiles from tens of thousands of individual cells. A major question in analyzing scRNA-seq data is how to account for the observed cell-to-cell variability. In this review, we provide an overview of scRNA-seq protocols, computational approaches for dissecting cellular heterogeneity, and future directions of single-cell transcriptomic analysis.

Application of Next Generation Sequencing to Investigate Microbiome in the Livestock Sector (Next Generation Sequencing을 통한 미생물 군집 분석의 축산분야 활용)

  • Kim, Minseok;Baek, Youlchang;Oh, Young Kyoon
    • Journal of Animal Environmental Science
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    • v.21 no.3
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    • pp.93-98
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    • 2015
  • The objective of this study was to review application of next-generation sequencing (NGS) to investigate microbiome in the livestock sector. Since the 16S rRNA gene is used as a phylogenetic marker, unculturable members of microbiome in nature or managed environments have been investigated using the NGS technique based on 16S rRNA genes. However, few NGS studies have been conducted to investigate microbiome in the livestock sector. The 16S rRNA gene sequences obtained from NGS are classified to microbial taxa against the 16S rRNA gene reference database such as RDP, Greengenes and Silva databases. The sequences also are clustered into species-level OTUs at 97% sequence similarity. Microbiome similarity among treatment groups is visualized using principal coordinates analysis, while microbiome shared among treatment groups is visualized using a venn diagram. The use of the NGS technique will contribute to elucidating roles of microbiome in the livestock sector.

Studies on the Oranization and Expression of tRNA Genes in Aspergillus nidulans (V) The Molecular Structure of $tRNA^{Arg}$ in Aspergillus nidulans (Aspergillus nidulans의 tRNA유전자의 구조와 발현에 관한 연구 V Aspergillus nidulansd의 $tRNA^{Arg}$ 분자구조)

  • 이병재;강현삼
    • Korean Journal of Microbiology
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    • v.24 no.2
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    • pp.79-85
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    • 1986
  • We have determined the sequence of $tRNA^{Arg}$ of A. nidulans partially by enzymatic rapid RNA sequencing technique. The sequence was 5'GGCCGGCUGGCCCAAXUGGCAAGGXUCUGAXUACGAAXCAGGAGAUUGCACXXXXXGAGCXXUXXGUCGGUCACCA3' The cloverleaf structure was made from above data. As a result, the anticodon sequence was identified as ACG. This result was confirmed with charging test. The complete sequence was proposed by supplementing the DNA sequence to and by assigning the position of minor bases to this RNA sequence.

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Recent next-generation sequencing and bioinformatic analysis methods for food microbiome research (식품 미생물 균총 연구를 위한 최신 마이크로바이옴 분석 기술)

  • Kwon, Joon-Gi;Kim, Seon-Kyun;Lee, Ju-Hoon
    • Food Science and Industry
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    • v.52 no.3
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    • pp.220-228
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    • 2019
  • Rapid development of next-generation sequencing (NGS) technology is available to study microbes in genomic level. This NGS has been widely used in DNA/RNA sequencing for genome sequencing, metagenomics, and transcriptomics. The food microbiology area could be categorized into three groups. Food microbes including probiotics and food-borne pathogens are studied in genomic level using NGS for microbial genomics. While food fermentation or food spoilage are more complicated, their genomic study needs to be done with metagenomics using NGS for compositional analysis. Furthermore, because microbial response in food environments are also important to understand their roles in food fermentation or spoilage, pattern analysis of RNA expression in the specific food microbe is conducted using RNA-Seq. These microbial genomics, metagenomics, and transcriptomics for food fermentation and spoilage would extend our knowledge on effective utilization of fermenting bacteria for health promotion as well as efficient control of food-borne pathogens for food safety.

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SNP Discovery from Transcriptome of Cashmere Goat Skin

  • Wang, Lele;Zhang, Yanjun;Zhao, Meng;Wang, Ruijun;Su, Rui;Li, Jinquan
    • Asian-Australasian Journal of Animal Sciences
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    • v.28 no.9
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    • pp.1235-1243
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    • 2015
  • The goat Capra hircus is one of several economically important livestock in China. Advances in molecular genetics have led to the identification of several single nucleotide variation markers associated with genes affecting economic traits. Validation of single nucleotide variations in a whole-transcriptome sequencing is critical for understanding the information of molecular genetics. In this paper, we aim to develop a large amount of convinced single nucleotide polymorphisms (SNPs) for Cashmere goat through transcriptome sequencing. In this study, the transcriptomes of Cashmere goat skin at four stages were measured using RNA-sequencing and 90% to 92% unique-mapped-reads were obtained from total-mapped-reads. A total of 56,231 putative SNPs distributed among 10,057 genes were identified. The average minor allele frequency of total SNPs was 18%. GO and KEGG pathway analysis were conducted to analyze the genes containing SNPs. Our follow up biological validation revealed that 64% of SNPs were true SNPs. Our results show that RNA-sequencing is a fast and efficient method for identification of a large number of SNPs. This work provides significant genetic resources for further research on Cashmere goats, especially for the high density linkage map construction and genome-wide association studies.

Microbial Community Analysis using RDP II (Ribosomal Database Project II):Methods, Tools and New Advances

  • Cardenas, Erick;Cole, James R.;Tiedje, James M.;Park, Joon-Hong
    • Environmental Engineering Research
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    • v.14 no.1
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    • pp.3-9
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    • 2009
  • Microorganisms play an important role in the geochemical cycles, industry, environmental cleanup, and biotechnology among other fields. Given the high microbial diversity, identification of the microorganism is essential in understanding and managing the processes. One of the most popular and powerful method for microbial identification is comparative 16S rRNA gene analysis. Due to the highly conserved nature of this essential gene, sequencing and later comparison of it against known rRNA databases can provide assignment of the bacteria into the taxonomy, and the identity of its closest relatives. Isolation and sequencing of 16S rRNA genes directly from natural environments (either from DNA or RNA) can also be used to study the structure of the whole microbial community. Nowadays, novel sequencing technologies with massive outputs are giving researchers worldwide the chance to study the microbial world with a depth that was previously too expensive to achieve. In this article we describe commonly used research approaches for the study of individual microorganisms and microbial communities using the tools provided by Ribosomal Database Project website.