Earticle

본 연구에서는 광주광역시 동림동 유적의 저습지에서 출토된 3점의 활엽수 수침고목재 (동림 1, 동림 2, 동림 3)를 대상으로 미생물 분해에 따른 해부학적, 화학적 특성 변화를 파악하 고 수침고목재에 존재하는 미생물 군집을 조사하였다. 동림 1−3의 최대함수율은 건전재의 최 대함수율보다 6∼8배 정도 높았으며 매장 기간과의 상관관계는 나타나지 않았다. 화학분석 결 과 동림 1−3의 회분 및 온수추출물 함량은 건전재에 비해 증가한 반면 유기용매 추출물 함량 은 감소하는 경향을 나타내었다. 아울러 홀로셀룰로오스 함량은 크게 감소하고 리그닌 함량은 상대적으로 크게 증가하였다. 현미경 관찰 결과 동림 1−3 모두에서 침식형 세균과 연부후균에 의한 피해가 관찰되었다. 차세대염기서열분석 기법을 통해 동림 2와 3의 미생물 군집을 분석한 결과 두 시료 모두에서 자낭균문(98∼99%)이 진균 군집(fungal community)의 대부분을 차지하 였고, 세균 군집(bacterial community)에서는 Proteobacteria문(34∼55%)이 가장 우세한 군집으로 검출되었다. 그러나 전체적인 미생물 군집 구조와 다양성은 동림 2와 3 사이에 현저한 차이를 보였다.

Anatomical and chemical changes due to microbial decay and microbial communities in three waterlogged archaeological woods, namely Dongnim 1, Dongnim 2, and Dongnim 3, excavated from the wetland of the Dongnim-dong archaeological site in Gwangju, were investigated. Dongnim 1−3 exhibited a maximum moisture content approximately 6 to 8 times higher than that of undecayed recent wood, and there was no correlation with the burial period. Chemical analysis revealed an increase in ash and hot water extractive content in Dongnim 1−3 compared to undecayed recent wood, while the solvent extractive content tended to decrease. Additionally, there was a significant decrease in holocellulose content with a relative increase in lignin concentration in Dongnim 1−3. Microscopic observations showed erosion bacteria decay and soft rot decay in Dongnim 1−3. The microbial community analysis of Dongnim 2 and 3 revealed the dominance of Ascomycota (98% to 99%) in the fungal community and Proteobacteria (34% to 55%) as the predominant bacterial phylum. Nonetheless, significant differences in microbial community structure and diversity were observed between Dongnim 2 and Dongnim 3.

Vanishing white matter (VWM) disease is an autosomal recessive disorder that affects the central nervous system of a patient, and is caused by the development of pathogenic mutations in any of the EIF2B1-5 genes. Any dysfunction of the EIF2B1-5 gene encoded eIF2B causes stress-provoked episodic rapid neurological deterioration in the patient, followed by a chronic progressive disease course. We present the case of a patient with an infantile-onset VWM with the pre-described specific clinical course, subsequent neurological aggravation induced by each viral infection, and the noted consequent progression into a comatose state. Although the initial brain magnetic resonance imaging did not reveal specific pathognomonic signs of VWM to distinguish it from other types of demyelinating leukodystrophy, the next-generation sequencing studies identified heterozygous missense variants in EIF2B3, including a novel variant in exon 7 (C706G), as well as a 0.008% frequency reported variant in exon 2 (T89C). Hence, the characteristic of unbiased genomic sequencing can clinically affect patient care and decision-making, especially in terms of the consideration of genetic disorders such as leukoencephalopathy in pediatric patients.

The transcriptomes of four ginseng accessions such as Cheonryang (Korean ginseng cultivar), Yunpoong(Korean ginseng cultivar), G03080 (breeding line of Korean ginseng), and P. quinquefolius (American ginseng) was characterized. As a result of sequencing, total lengths of the reads in each sample were 156.42 Mb (Cheonryang cultivar), 161.95Mb (Yunpoong cultivar), 165.07 Mb (G03080 breeding line), and 166.48 Mb (P. quinquefolius). Using a BLAST searchagainst the Phytozome databases with an arbitrary expectation value of 1E-10, over 20,000 unigenes were functionally annotatedand classified using DAVID software, and were found in response to external stress in the G03080 breeding line, as wellas in the Cheonryang cultivar, which was associated with the ion binding term. Finally, unigenes related to transmembranetransporter activity were observed in Cheonryang and P. quinquefolius, which involves controlling osmotic pressure and turgorpressure within the cell. The expression patterns were analyzed to identify dehydrin family genes that were abundantlydetected in the Cheonryang cultivar and the G03080 breeding line. In addition, the Yunpoong cultivar and P. quinquefoliusaccession had higher expression of heat shock proteins expressed in Ricinus communis. These results will be a valuableresource for understanding the structure and function of the ginseng transcriptomes.

The cloud environment makes it possible to analyze large data sets in a scalable computing infrastructure. In the bioinformatics field, the applications are composed of the complex workflow tasks, which require huge data storage as well as a computing-intensive parallel workload. Many approaches have been introduced in distributed solutions. However, they focus on static resource provisioning with a batch-processing scheme in a local computing farm and data storage. In the case of a large-scale workflow system, it is inevitable and valuable to outsource the entire or a part of their tasks to public clouds for reducing resource costs. The problems, however, occurred at the transfer time for huge dataset as well as there being an unbalanced completion time of different problem sizes. In this paper, we propose an adaptive resource-provisioning scheme that includes run-time data distribution and collection services for hiding the data transfer time. The proposed adaptive resource-provisioning scheme optimizes the allocation ratio of computing elements to the different datasets in order to minimize the total makespan under resource constraints. We conducted the experiments with a well-known sequence alignment algorithm and the results showed that the proposed scheme is efficient for the cloud environment.

본 연구는 콩과 식물인 비수리의 유용유전자를 NGS (Next Generation Sequencing)와 분자생물학의 융합적인 연구를 통해 분리하고 가능성을 알아보고자 시행하였다. 비수리는 자원식물이지만 많은 유용물질을 가지고 있다. 특히 항당뇨 기능을 하는 D-pinitol을 많이 함유하고 있는데 아직까지 비수리에서 D-piniol의 생합성에 관련된 유전자가 분리 되지 않았다. 비수리에 비생물학적 스트레스(가뭄)를 처리하고 처리하지 않은 대조군과 같이 total RNA를 추출한 후에 library를 만들어 NGS를 실시하였다. 이를 통해 D-pinitol 생합성에 관련된 유전자들을 분리하여 in silico 상에서 염기서열을 확인하였다. 이를 뒷받침하기 위해 Blast 프로그램을 사용하여 D-pinitol 생함성에 관여하는 ononitol epimerase를 확인하였고 in vitro 상에서도 RT-PCR을 통해 유전자 발현이 증가됨을 확인함으로써 융합적 연구를 통해 유전자를 찾고 분리하여 발현양상을 확인하였다.

This study was carried out to investigate the possibility of isolating the useful gene of soybean plant, anthocyanin, through NGS (Next Generation Sequencing) and molecular biology experiments. Lespedeza cuneata. G. don is a resource plant but has many useful materials. Especially, D-pinitol, which has anti-diabetic function, is contained in a large amount. However, the gene related to the biosynthesis of D-piniol has not been isolated in the non-spermatid. Lespedeza cuneata. G. don was treated with abiotic stress (drought), total RNA was extracted, and a library was constructed to perform NGS. In this way, the genes involved in D-pinitol biosynthesis were isolated and sequenced in silico. In order to support this, ononitol epimerase involved in D-pinitol amplification was identified using the Blast program and RT-PCR confirmed the increased gene expression in vitro, and the gene was isolated and identified by convergence study.

Background: Adenophora triphylla var. japonica (Regel) H. Hara shows vegetative growth with radical leaves during the first year and shows reproductive growth with cauline leaves and bolting during the second year. In addition, the shape of the plant varies within the same species. For this reason, there are limitations to classifying the species by visual examination. However, there is not sufficient genetic information or molecular tools to analyze the genetic diversity of the plant. Methods and Results: Approximately 34.59 Gbp of raw data containing 342,487,502 reads was obtained from next generation sequencing (NGS) and these reads were assembled into 357,211 scaffolds. A total of 84,106 simple sequence repeat (SSR) regions were identified and 14,133 primer sets were designed. From the designed primer sets, 95 were randomly selected and were applied to the genomic DNA which was extracted from five plants and pooled. Thirty-nine primer sets showing more than two bands were finally selected as SSR markers, and were used for the genetic relationship analysis. Conclusions: The 39 novel SSR markers developed in this study could be used for the genetic diversity analysis, variety identification, new variety development and molecular breeding of A. triphylla

Next generation sequencing (NGS) has been carried out for a number of purposes in medicine, anthropology, population and forensic genetics. It can produce massive sequencing data, and is expected to become and alternative or an additional approach to overcome the limitation of capillary electrophoresis(CE)- based forensic STR typing, especially in the analyses of mixed samples. However, there has been no available multiplex PCR system that is optimized for NGS analysis of STRs. In this regard, we constructed a multiplex PCR system for NGS analysis STRs, which composed of 4 markers of commonly used commercial kits(vWA, D7S820, D21S11 and D8S1179). Amplicons were designed to have a size ranging about 200 bp which is compatible with the read length of currently available NGS platforms. To validated the multiplex PCR system, PCR products were generated from single-source samples and the mixed DNA samples in a varying ratios, and subsequent barcodeed library was prepared and sequenced on a benchtop sequencer, GS Junior Sequencer(Roche). STR typing results obtained from NGS analysis were consistent with those from CE-based analyses both for single-source samples and mixed samples. As a result, STR types are not easy to distinguish major and minor contributor in mixed DNA samples. In contrast, NGS sequencing was able to differentiate among them by DNA sequence variation. This results suggest that NGS can be one of the good method to indicate who is the contributor in mixed DNA which was originated from crime scene evidence. In addition, we examined DNA sequence of 6 microvariants in D21S11 locus using NGS to find out particular character of D21S21 microvariants. They showed mutations and sequence variations in the repeat region of D21S11 short tandem repeat (STR) loci.

본 연구에서 사용된 차세대 염기서열 분석 기반의 비교시험은 Applied Biosystems社의 3130 xl과 Roche社의 GS junior (454 GS FLX) 염기서열 분석 장비를 이용하여 60년 이상 오래된 유해 100구의 mtDNA 염기서열 분석을 수행하였다. 그 결과, GS junior가 95%에 해당하는 mtDNA 고변이 좌위에서 더 높은 민감도와 식별력을 갖는 것으로 나타났다. 또한, 전반적인 염기서열 분석 결과의 base-calling 오 류는 ‘random sampling’ 오류를 감안하더라도 높은 coverage 및 depth에 의해 상대적으로 낮은 수치를 나 타냈다. GS junior는 상대적으로 많은 리드를 확보할 수 있기 때문에 단일 염기 변이 분석 결과에서 3130 xl에 비해 6% 더 많은 염기 변이를 찾은 것으로 나타났다. 그러나 HV2의 309와 315에서 C insertion 으로 6 량체 이상 늘어나게 되면 poly C-stretch 현상이 발생하여 분석 성공률이 낮아진다. GS junior에서 는 309와 315의 분석 성공률이 3130 xl에 비해 25%와 70%로 나타나 상대적으로 낮게 분석되었다.

To comparison these systems for NGS platform, we analyzed mtDNA sequence generated by the Applied Biosystems 3130 xl and Roche GS junior (454 GS FLX) technologies for the over 60 years skeletal remains of 100 individuals. Consequently, a comparison of the base calls of overlapping ABI 3130 xl Sangersequence generated for the same samples showed that the NGS platforms all have high sensitivity, identifying 95% of mtDNA hypervariable region. At high coverage, depth base-calling error are systematic, resulting from local sequence contexts; as the coverage is lowered additional 'random sampling' errors in base-calling occur. GS junior because you can gain a lot of re from a single variant analysis showed that 6% more as compared to the 3130 xl variant is found. However, the C insertion occurs of HV2 in the 309 and 315, increases more than 6 mer poly C-stretch phenomenon occurs, it becomes very difficult to analyze. Analysis of GS junior analyze the success rate in the 309 and 315 is shown as 25% and 70% compared to the 3130 xl relatively low.

The predator-prey interaction in freshwater ecosystems is a crucial area in the ecological study field and one of example to find such interaction is to investigate stomach contents. However, traditional method through visual inspection often induce misidentification, as it depends critically on intactness of physically visible data. In this study, we utilized Next-Generations Sequencing (NGS) technology to test the applicability stomach content analysis and overcome such limitation. NGS was applied to analyze the stomach contents of the Hemibarbus labeo, Tachysurus fulvidraco, and Plecoglossus altivelis collected in the lower part of Nakdong River. As a result, T. fulvidraco had a higher number of Animalia operational taxonomic units (OTUs) intake rate than H. labeo. At the same time, P. altivelis had higher number of Plantae OTUs intake rate than T. fulvidraco and higher Protozoa OTUs intake rate than H. labeo respectively. Therefore, NGS technology application enable to overcome traditional method's limitation and discover hidden interspecific interaction which can further be used in appropriate habitat assessment.

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.

법유전학에서 개인의 신원확인을 위한 STR 프로필 분석이 불가한 경우, DNA를 이용한 외형추정특성을 이용하여 개인에 대한 정보를 얻을 수 있다. 최근 눈동자, 머리카락, 피부 색과 같은 외형추정특성을 확인하는 방법들이 연구되고 있지만, 이러한 외형추정특성 정보만 가지고는 한국을 비롯한 동아시아 지역에서 적용하기에는 한계가 있다. 본 연구에서는 개인의 외형과 관련된 표현형을 수사정보로서 활용하기 위해 눈 모양, 머리카락 굵기, 피부 색 뿐만 아니라 탈모, 체형, 고도근시, 얼굴모양, 여드름, 행동습관과 관련된 SNP를 탐색하였다. 이들 표현형과 관련된 50개의 SNP를 선정하여 한 번에 증폭할 수 있는 targeted amplicon NGS 방식의 multiplex PCR 패널을 개발하였다. 실험 결과 14개 샘플에서 50개 SNP의 대립유전자 유형과 빈도를 확인할 수 있었다. 향후 본 패널을 가지고 더 많은 샘플을 이용하여 유전형과 표현형 간 연관성 확인 및 결과 해석 방법을 분석할 예정이다.

In forensic genetics, when it is not possible to confirm an individual's identity through STR profile analysis, additional information about the individual can be obtained using DNA-based phenotypic traits estimation. Recently, various researches have been conducted on methods to determine externally visible characteristics (EVC) such as eyes, hair, and skin color. However, relying solely on such phenotypic traits information has limitations for application in East Asian regions, including Korea. In this study, in order to utilize EVC related to an individual's appearance as investigative information, SNPs related to eye shape, hair thickness, skin color, as well as baldness, body type, high myopia, facial shape, acne, and behavioral habits were explored. A total of 50 SNPs were selected, and a targeted amplicon NGS panel capable of amplifying them all at once was developed. Experimental results confirmed the allelic types and frequencies of the 50 SNPs in 14 samples. We plan to use this panel to investigate the correlation between genotype and phenotype using various samples, and to develop methods for interpreting the results.

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.

The evolving landscape of precision oncology underscores the pivotal shift from morphological diagnosis to treatment decisions driven by molecular profiling. Recent guidelines from the European Society for Medical Oncology recomend the use of next-generation sequencing (NGS) across a broader range of cancers, reflecting its superior efficiency and clinical value. NGS not only updates oncology testing by offering quicker, sample-friendly, and sensitive analysis but also reduces the need for multiple individual tests. Cytology samples, often obtained through less invasive methods, can yield high-quality genetic material suitable for molecular analysis. This article focuses on optimizing the use of cytology samples in NGS, and outlines their potential benefits in identifying actionable molecular alterations for targeted therapies across various solid tumors. It also addresses the need for validation studies and the strategies to incorporate or combine different types of samples into routine clinical practice. Integrating cytological and liquid biopsies into routine clinical practice, alongside conventional tissue biopsies, offers a comprehensive approach to tumor genotyping, early disease detection, and monitoring of therapeutic responses across various solid tumor types. For comprehensive biomarker characterization, all patient specimens, although limited, is always valuable.

In countries with FMD vaccination, as in Korea, typical clinical signs do not appear, and even in FMD positive cases, it is difficult to isolate the FMDV or obtain whole genome sequence. To overcome this problem, more rapid and simple NGS system is required to control FMD in Korea. FMDV (O/Boeun/ SKR/2017) RNA was extracted and sequenced using Ion Torrent's bench-top sequencer with amplicon panel with optimized bioinformatics pipelines. The whole genome sequencing of raw data generated data of 1,839,864 (mean read length 283 bp) reads comprising a total of 521,641,058 (≥Q20 475,327,721). Compared with FMDV (GenBank accession No. MG983730), the FMDV sequences in this study showed 99.83% nucleotide identity. Further study is needed to identify these differences. In this study, fast and robust methods for benchtop next generation sequencing (NGS) system was developed for analysis of Foot-and-mouth disease virus (FMDV) whole genome sequences.

PacBio's long-read sequencing technologies can be successfully used for a complete bacterial genome assembly using recently developed non-hybrid assemblers in the absence of second-generation, high-quality short reads. However, standardized procedures that take into account multiple pre-existing second-generation sequencing platforms are scarce. In addition to Illumina HiSeq and Ion Torrent PGM-based genome sequencing results derived from previous studies, we generated further sequencing data, including from the PacBio RS II platform, and applied various bioinformatics tools to obtain complete genome assemblies for five bacterial strains. Our approach revealed that the hierarchical genome assembly process (HGAP) non-hybrid assembler resulted in nearly complete assemblies at a moderate coverage of ~75x, but that different versions produced non-compatible results requiring post processing. The other two platforms further improved the PacBio assembly through scaffolding and a final error correction.

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.

Purpose: Human milk microbiota can significantly influence children's health. This study aimed to investigate the composition and diversity of the bacterial microbiota in donated human milk (DHM) and analyze the possible influencing factors. Methods: Archival samples from single-donor pools of raw DHM expressed from 88 different donors within the first six months postpartum were included. Donor-, infant-, and milk-related data were collected and analyzed using descriptive and comparative statistical methods. The hypervariable region V1-V3 of the bacterial 16S ribosomal ribonucleic acid gene was sequenced using the Illumina MiSeq platform to characterize the microbial profile of the donated milk. Results: Staphylococcus was the most abundant genus in all analyzed samples. The genus Lactobacillus was also present in all samples, with relative abundances ranging from 0.06-62%. Bacteria of the genus Bifidobacterium were present in 62 (70% n=62) of the samples. A statistically significant difference in Lactobacillus abundance was observed based on the storage container type before pasteurization (p=0.015, median 0.804 in bottles vs. 0.289 in bags). Alpha diversity was higher in milk samples from exclusively breastfed infants, and in the samples that were expressed using manual techniques. Conclusion: This study provides evidence of the potential influence of donor infantrelated factors and milk characteristics on the composition and alpha diversity of bacterial microbiota in DHM. Notably, previous studies utilizing high-throughput sequencing technology to examine the human milk microbiota have not focused on pooled DHM samples.

Molecular markers have been widely applied in population genetics, diagnostic taxonomy, and genetic mapping, and they can also be used for classifying varieties of Italian ryegrass during field selection. In this study, genome-wide sequence information was generated for 10 Italian ryegrass cultivars (40 samples), including 'Kowinearly' (KW), using next-generation sequencing (NGS). Single nucleotide polymorphism (SNP) analysis revealed that only three SNP loci were sufficient to distinguish KW from the other cultivars. Furthermore, 21 alternative barcode sets, each consisting of three SNPs, were identified. These SNP barcode sets provide a reliable criterion for cultivar discrimination in Italian ryegrass and can contribute to the protection of domestic varieties and the advancement of the forage industry in Korea. More broadly, the development of distinguishing markers across Italian ryegrass cultivars will enhance genetic resource identification and support the breeding of high-quality new varieties.