High-throughput identification of chrysanthemum gene function and expression: An overview and an effective proposition

Toan Khac Nguyen,Jin Hee Lim 한국식물생명공학회 2021 JOURNAL OF PLANT BIOTECHNOLOGY Vol.48 No.3

Since whole-genome duplication (WGD) of diploid Chrysanthemum nankingense and de novo assembly whole-genome of C. seticuspe have been obtained, they have afforded to perceive the diversity evolution and gene discovery in the improved investigation of chrysanthemum breeding. The robust tools of high-throughput identification and analysis of gene function and expression produce their vast importance in chrysanthemum genomics. However, the gigantic genome size and heterozygosity are also mentioned as the major obstacles preventing the chrysanthemum breeding practices and functional genomics analysis. Nonetheless, some of technological contemporaries provide scientific efficient and promising solutions to diminish the drawbacks and investigate the high proficient methods for generous phenotyping data obtaining and system progress in future perspectives. This review provides valuable strategies for a broad overview about the high-throughput identification, and molecular analysis of gene function and expression in chrysanthemum. We also contribute the efficient proposition about specific protocols for considering chrysanthemum genes. In further perspective, the proper high-throughput identification will continue to advance rapidly and advertise the next generation in chrysanthemum breeding.

Characterization of microbial community composition and pathogens risk assessment in typical Italian-style salami by high-throughput sequencing technology

Xinhui Wang,Hongyang Ren,Yi Zhang 한국식품과학회 2018 Food Science and Biotechnology Vol.27 No.1

The structure of microbial communities in a typical Italian-style salami, including bacterial and fungal diversity, was investigated by high-throughput sequencing technology. A total of 6 phyla, 7 classes, 19 orders, 20 families and 28 genera were obtained from 16S rDNA sequences, and a total of 2 phyla, 4 classes, 4 orders, 5 families, 10 genera and 12 Species were obtained from 18S rDNA sequences. The core microbiota was composed of Staphylococcaceae, representing up to 97.52% of the total 16S rRNA, and Penicillium digitatum, accounting for 99.74% of the total classified 18S rRNA. Lactobacillales and Saccharomycetales were detected with a quite low proportion of 1.71 and 0.007%, respectively. This study contributes to the knowledge of the microbial diversity involved in salami and presents high-throughput sequencing as a useful tool to evaluate microbial diversity and monitor the food-borne pathogens in fermented sausage.

New Lung Cancer Panel for High-Throughput Targeted Resequencing

김은혜,김병철,이성훈,박종선,이규상,박종화 한국유전체학회 2014 Genomics & informatics Vol.12 No.2

We present a new next-generation sequencing-based method to identify somatic mutations of lung cancer. It is acomprehensive mutation profiling protocol to detect somatic mutations in 30 genes found frequently in lung adenocarcinoma. The total length of the target regions is 107 kb, and a capture assay was designed to cover 99% of it. This methodexhibited about 97% mean coverage at 30× sequencing depth and 42% average specificity when sequencing of more than3.25 Gb was carried out for the normal sample. We discovered 513 variations from targeted exome sequencing of lungcancer cells, which is 3.9-fold higher than in the normal sample. The variations in cancer cells included previously reportedsomatic mutations in the COSMIC database, such as variations in TP53, KRAS, and STK11 of sample H-23 and in EGFR ofsample H-1650, especially with more than 1,000× coverage. Among the somatic mutations, up to 91% of single nucleotidepolymorphisms from the two cancer samples were validated by DNA microarray-based genotyping. Our resultsdemonstrated the feasibility of high-throughput mutation profiling with lung adenocarcinoma samples, and the profilingmethod can be used as a robust and effective protocol for somatic variant screening.

Characterization of microbial community composition and pathogens risk assessment in typical Italian-style salami by high-throughput sequencing technology

Wang, Xinhui,Ren, Hongyang,Zhan, Yi 한국식품과학회 2018 Food Science and Biotechnology Vol.27 No.1

The structure of microbial communities in a typical Italian-style salami, including bacterial and fungal diversity, was investigated by high-throughput sequencing technology. A total of 6 phyla, 7 classes, 19 orders, 20 families and 28 genera were obtained from 16S rDNA sequences, and a total of 2 phyla, 4 classes, 4 orders, 5 families, 10 genera and 12 Species were obtained from 18S rDNA sequences. The core microbiota was composed of Staphylococcaceae, representing up to 97.52% of the total 16S rRNA, and Penicillium digitatum, accounting for 99.74% of the total classified 18S rRNA. Lactobacillales and Saccharomycetales were detected with a quite low proportion of 1.71 and 0.007%, respectively. This study contributes to the knowledge of the microbial diversity involved in salami and presents high-throughput sequencing as a useful tool to evaluate microbial diversity and monitor the food-borne pathogens in fermented sausage.

High-throughput part characterization tools with a long-read sequencing technique

Haseong KIM 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

It is known that the optimal performance of a metabolic pathway is achieved by the appropriate expression balance among genes. However, investigating each DNA part that controls the expression of the genes requires time-consuming cloning experiments along with high-cost equipment measuring single-cell fluoresce. This study presents a high-throughput part characterizing technique that increases cloning throughput by combinatorial DNA part assembly approach and quantifies the part characteristics with High-reproducibility plate based image analysis. The PCR based long-read sequencing technique allows us fast identification of part combinations and their genotypes regardless relatively low base-calling accuracy of the long-read sequencing technique. We confirmed the ratios of the part combinations were constantly remained in each assembly step and the fluorescent intensities of the colonies on plates were comparable to the single-cell level fluorescence from a high-end flowcytometry device. Quantitative values of twenty DNA parts (23 promoters and 23 RBSs) were successfully obtained in 1 week without any automated and expensive devices. We anticipate that this high-throughput and easy-to-use part characterization technique will contribute to increase the available parts in the synthetic biology community.

The Principles and Applications of High-Throughput Sequencing Technologies

이준영 한국발생생물학회 2023 발생과 생식 Vol.27 No.1

The advancement in high-throughput sequencing (HTS) technology has revolutionized the field of biology, including genomics, epigenomics, transcriptomics, and metagenomics. This technology has become a crucial tool in many areas of research, allowing scientists to generate vast amounts of genetic data at a much faster pace than traditional methods. With this increased speed and scale of data generation, researchers can now address critical questions and gain new insights into the inner workings of living organisms, as well as the underlying causes of various diseases. Although the first HTS technology have been introduced about two decades ago, it can still be challenging for those new to the field to understand and use effectively. This review aims to provide a comprehensive overview of commonly used HTS technologies these days and their applications in terms of genome sequencing, transcriptome, DNA methylation, DNA-protein interaction, chromatin accessibility, three-dimensional genome organization, and microbiome.

Trends in Next-Generation Sequencing and a New Era for Whole Genome Sequencing

박상태,김자영 대한배뇨장애요실금학회 2016 International Neurourology Journal Vol.20 No.S1

This article is a mini-review that provides a general overview for next-generation sequencing (NGS) and introduces one of the most popular NGS applications, whole genome sequencing (WGS), developed from the expansion of human genomics. NGS technology has brought massively high throughput sequencing data to bear on research questions, enabling a new era of genomic research. Development of bioinformatic software for NGS has provided more opportunities for researchers to use various applications in genomic fields. De novo genome assembly and large scale DNA resequencing to understand genomic variations are popular genomic research tools for processing a tremendous amount of data at low cost. Studies on transcriptomes are now available, from previous-hybridization based microarray methods. Epigenetic studies are also available with NGS applications such as whole genome methylation sequencing and chromatin immunoprecipitation followed by sequencing. Human genetics has faced a new paradigm of research and medical genomics by sequencing technologies since the Human Genome Project. The trend of NGS technologies in human genomics has brought a new era of WGS by enabling the building of human genomes databases and providing appropriate human reference genomes, which is a necessary component of personalized medicine and precision medicine.

New Lung Cancer Panel for High-Throughput Targeted Resequencing

Kim, Eun-Hye,Lee, Sunghoon,Park, Jongsun,Lee, Kyusang,Bhak, Jong,Kim, Byung Chul Korea Genome Organization 2014 Genomics & informatics Vol.12 No.2

We present a new next-generation sequencing-based method to identify somatic mutations of lung cancer. It is a comprehensive mutation profiling protocol to detect somatic mutations in 30 genes found frequently in lung adenocarcinoma. The total length of the target regions is 107 kb, and a capture assay was designed to cover 99% of it. This method exhibited about 97% mean coverage at $30{\times}$ sequencing depth and 42% average specificity when sequencing of more than 3.25 Gb was carried out for the normal sample. We discovered 513 variations from targeted exome sequencing of lung cancer cells, which is 3.9-fold higher than in the normal sample. The variations in cancer cells included previously reported somatic mutations in the COSMIC database, such as variations in TP53, KRAS, and STK11 of sample H-23 and in EGFR of sample H-1650, especially with more than $1,000{\times}$ coverage. Among the somatic mutations, up to 91% of single nucleotide polymorphisms from the two cancer samples were validated by DNA microarray-based genotyping. Our results demonstrated the feasibility of high-throughput mutation profiling with lung adenocarcinoma samples, and the profiling method can be used as a robust and effective protocol for somatic variant screening.

Whole Exome Sequencing of a Patient with Duchenne Muscular Dystrophy

최봉석(Bong Seok Choi),황수경(Su-Kyeong Hwang) 대한소아신경학회 2014 대한소아신경학회지 Vol.22 No.1

듀센형 근이영양증은 소아에서 가장 흔하고 치명적인 유전성 근육질환으로, 디스트로핀 유전자의 변이로 인해 발생한다. 디스트로핀 유전자의 결실이나 중복에 의해 생기는 경우가 많으므로 MLPA나 aCGH가 진단을 위해 많이 쓰인다. 그러나 환자의 1/3 에서는 변이가 발견되지 않는데, 이럴 경우 direct sequencing을 통해 MLPA나 aCGH에서 확인되지 않는 미세한 결실, 중복, 또는 point mutation이 있는지 확인하게 된다. 그러나 DMD 유전자는 크기가 매우 크기 때문에 direct sequencing을 하기에는 시간과 비용이 엄청나게 소모되며 최종 결과도 확정적이지 않을 가능성이 있다. 우리는 MLPA에서 변이가 발견되지 않은 진행성 근이영양증 환자를 대상으로 whole exome sequencing을 시행하였는데, 디스트로핀 유전자의 frame shift mutation이 발견되었다. 환자의 어머니를 대상으로 시행한 direct sequencing에서도 환자와 같은 변이가 발견되었다. Whole exome sequencing은 MLPA에서 변이가 발견되지 않는 듀센형 근이영양증 환자의 새로운 진단 도구로 훌륭한 역할을 할 수 있을 것으로 생각된다. Duchenne muscular dystrophy (DMD) is the most common and lethal dystrophy in childhood, caused by mutations in the dystrophin (DMD) gene. Multiplex ligation dependent probe amplification (MLPA) or array comparative genome hybridization (aCGH) is widely used as an initial molecular diagnostic tool. If no deletions or duplications are found in MLPA or aCGH, the samples must be subjected to a second test of direct sequencing. Direct sequencing of the DMD gene, however, is timeconsuming, high-cost, and can be inconclusive. Here, we performed whole exome sequencing on a patient with progressive muscle weakness whose MLPA result was negative; the result revealed a rare frame shift mutation. Direct sequencing on the patient's mother showed the same mutation. Whole exome sequencing can be a new diagnostic routine for DMD patients with negative MLPA3

CASPER: context-aware scheme for paired-end reads from high-throughput amplicon sequencing

Kwon, Sunyoung,Lee, Byunghan,Yoon, Sungroh BioMed Central 2014 BMC bioinformatics Vol.15 No.suppl9

<P>Merging the forward and reverse reads from paired-end sequencing is a critical task that can significantly improve the performance of downstream tasks, such as genome assembly and mapping, by providing them with virtually elongated reads. However, due to the inherent limitations of most paired-end sequencers, the chance of observing erroneous bases grows rapidly as the end of a read is approached, which becomes a critical hurdle for accurately merging paired-end reads. Although there exist several sophisticated approaches to this problem, their performance in terms of quality of merging often remains unsatisfactory. To address this issue, here we present a <B>c</B>ontext-<B>a</B>ware scheme for <B>p</B>aired-<B>e</B>nd <B>r</B>eads (CASPER): a computational method to rapidly and robustly merge overlapping paired-end reads. Being particularly well suited to amplicon sequencing applications, CASPER is thoroughly tested with both simulated and real high-throughput amplicon sequencing data. According to our experimental results, CASPER significantly outperforms existing state-of-the art paired-end merging tools in terms of accuracy and robustness. CASPER also exploits the parallelism in the task of paired-end merging and effectively speeds up by multithreading. CASPER is freely available for academic use at http://best.snu.ac.kr/casper.</P>