Whole-Genome and Transcriptome Sequencing Identified NOTCH2 and HES1 as Potential Markers of Response to Imatinib in Desmoid Tumor (Aggressive Fibromatosis): A Phase II Trial Study

권준하,이준형,이영한,이지연,안진희,김세현,김승현,김태일,윤금희,박영석,김정은,이규상,최정균,김효송 대한암학회 2022 Cancer Research and Treatment Vol.54 No.4

Purpose Desmoid tumor, also known as aggressive fibromatosis, is well-characterized by abnormal Wnt/β-catenin signaling. Various therapeutic options, including imatinib, are available to treat desmoid tumor. However, the molecular mechanism of why imatinib works remains unclear. Here, we describe potential roles of NOTCH2 and HES1 in clinical response to imatinib at genome and transcriptome levels. Materials and Methods We identified somatic mutations in coding and noncoding regions via whole-genome sequencing. To validate the genetic interaction with expression level in desmoid-tumor condition, we utilized large-scale whole-genome sequencing and transcriptome datasets from the Pan-Cancer Analysis of Whole Genomes project. RNA-sequencing was performed using prospective and retrospective cohort samples to evaluate the expressional relevance with clinical response. Results Among 20 patients, four (20%) had a partial response and 14 (66.7%) had stable disease, 11 of which continued for ≥ 1 year. With gene-wise functional analyses, we detected a significant correlation between recurrent NOTCH2 noncoding mutations and clinical response to imatinib. Based on Pan-Cancer Analysis of Whole Genomes data analyses, NOTCH2 mutations affect expression levels particularly in the presence of CTNNB1 missense mutations. By analyzing RNA-sequencing with additional desmoid tumor samples, we found that NOTCH2 expression was significantly correlated with HES1 expression. Interestingly, NOTCH2 had no statistical power to discriminate between responders and non-responders. Instead, HES1 was differentially expressed with statistical significance between responders and non-responders. Conclusion Imatinib was effective and well tolerated for advanced desmoid tumor treatment. Our results show that HES1, regulated by NOTCH2, as an indicator of sensitivity to imatinib, and an important therapeutic consideration for desmoid tumor. Purpose Desmoid tumor, also known as aggressive fibromatosis, is well-characterized by abnormal Wnt/β-catenin signaling. Various therapeutic options, including imatinib, are available to treat desmoid tumor. However, the molecular mechanism of why imatinib works remains unclear. Here, we describe potential roles of <i>NOTCH2</i> and <i>HES1</i> in clinical response to imatinib at genome and transcriptome levels.Materials and Methods We identified somatic mutations in coding and noncoding regions via whole-genome sequencing. To validate the genetic interaction with expression level in desmoid-tumor condition, we utilized large-scale whole-genome sequencing and transcriptome datasets from the Pan-Cancer Analysis of Whole Genomes project. RNA-sequencing was performed using prospective and retrospective cohort samples to evaluate the expressional relevance with clinical response.Results Among 20 patients, four (20%) had a partial response and 14 (66.7%) had stable disease, 11 of which continued for ≥ 1 year. With gene-wise functional analyses, we detected a significant correlation between recurrent <i>NOTCH2</i> noncoding mutations and clinical response to imatinib. Based on Pan-Cancer Analysis of Whole Genomes data analyses, <i>NOTCH2</i> mutations affect expression levels particularly in the presence of CTNNB1 missense mutations. By analyzing RNA-sequencing with additional desmoid tumor samples, we found that <i>NOTCH2</i> expression was significantly correlated with <i>HES1</i> expression. Interestingly, <i>NOTCH2</i> had no statistical power to discriminate between responders and non-responders. Instead, <i>HES1</i> was differentially expressed with statistical significance between responders and non-responders.Conclusion Imatinib was effective and well tolerated for advanced desmoid tumor treatment. Our results show that <i>HES1</i>, regulated by <i>NOTCH2</i>, as an indicator of sensitivity to imatinib, and an important therapeutic consideration for desmoid tumor.

Rapid Whole-genome Sequencing of Zika Viruses using Direct RNA Sequencing

Jung Heon Kim,Ji Yeon Kim,구본상,Hanseul Oh,홍정주,EUNG-SOO HWANG 대한미생물학회 2019 Journal of Bacteriology and Virology Vol.49 No.3

Zika virus (ZIKV) is one of the pathogens which is transmitted world widely, but there are no effective drugs and vaccines. Whole genome sequencing (WGS) of viruses could be applied to viral pathogen characterization, diagnosis, molecular surveillance, and even finding novel pathogens. We established an improved method using direct RNA sequencing with Nanopore technology to obtain WGS of ZIKV, after adding poly (A) tails to viral RNA. This established method does not require specific primers, complimentary DNA (cDNA) synthesis, and polymerase chain reaction (PCR)-based enrichment, resulting in the reduction of biases as well as of the ability to find novel RNA viruses. Nanopore technology also allows to read long sequences. It makes WGS easier and faster with long-read assembly. In this study, we obtained WGS of two strains of ZIKV following the established protocol. The sequenced reads resulted in 99% and 100% genome coverage with 63.5X and 21,136X, for the ZIKV PRVABC59 and MR 766 strains, respectively. The sequence identities of the ZIKV PRVABC59 and MR 766 strains for each reference genomes were 98.76% and 99.72%, respectively. We also found that the maximum length of reads was 10,311 bp which is almost the whole genome size of ZIKV. These long-reads could make overall structure of whole genome easily, and WGS faster and easier. The protocol in this study could provide rapid and efficient WGS that could be applied to study the biology of RNA viruses including identification, characterization, and global surveillance.

Whole genome sequencing of foot-and-mouth disease virus using benchtop next generation sequencing (NGS) system

Sung-Hyun Moon,오연수,Dongseob Tark,조호성 한국동물위생학회 2019 한국동물위생학회지 (KOJVS) Vol.42 No.4

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 show-ed 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.

Sequencing and comparison of head louse whole genome sequences with those of body louse genome

Jae Soon Kang,Young-Jun Cho,Ju Hyeon Kim,Si Hyeock Lee 한국응용곤충학회 2011 한국응용곤충학회 학술대회논문집 Vol.2011 No.05

The differences in the immune response between body lice, Pediculus humanus humanus, and head lice, Pediculus humanus capitis, were regarded as primary factors determining their differential vector competence. To find any differences in genetic components in immune system between body and head lice, whole genome sequences of head lice were determined by both SBS [sequencing by synthesis, Illumina Genome Analyzer (Illumina-GA)] and pyrosequencing (Roche GS FLX), and compared with the reference genome sequences of body lice. The short DNA reads from Illumina-GA (an average mapping depth of 50-fold) were aligned first to the body louse reference genome, to which Roche GS FLX DNA reads (an average depth of 2.5-fold) were subsequently assembled to make up gaps between mapped consensus. Total consensus showed a size of 114 Mb and a coverage of 96% of the published body louse genome sequences. From this head louse genome sequences, a total of 12,651 genes were predicted and used for comparing with the 10,775 genes previously reported from the body louse genome. The homolog analysis identified 873 head louse-specific genes and 422 body lice-specific genes. Comparison of immune response genes between both louse species showed head lice have more number of immune-related genes than body lice. Head lice were determined to possess all of the 107 immune-related genes reported in the previous study (Kim et al., 2011), suggesting that there is no difference in genetic make-up in terms of the 107 immune-related genes between body and head lice.

Whole-genome sequence analysis through online web interfaces: a review

Gunasekara, A.W.A.C.W.R.,Rajapaksha, L.G.T.G.,Tung, T.L. Korea Genome Organization 2022 Genomics & informatics Vol.20 No.1

The recent development of whole-genome sequencing technologies paved the way for understanding the genomes of microorganisms. Every whole-genome sequencing (WGS) project requires a considerable cost and a massive effort to address the questions at hand. The final step of WGS is data analysis. The analysis of whole-genome sequence is dependent on highly sophisticated bioinformatics tools that the research personal have to buy. However, many laboratories and research institutions do not have the bioinformatics capabilities to analyze the genomic data and therefore, are unable to take maximum advantage of whole-genome sequencing. In this aspect, this study provides a guide for research personals on a set of bioinformatics tools available online that can be used to analyze whole-genome sequence data of bacterial genomes. The web interfaces described here have many advantages and, in most cases exempting the need for costly analysis tools and intensive computing resources.

Whole-genome Sequencing of Two Liver Tumors from One Patient

( Tae Hyung Kim ),( Soon Ho Um ),( Yeon Seok Seo ),( Seung Woon Park ),( Han Ah Lee ),( Sang Jung Park ),( Dong-sik Kim ),( Young Dong Yu ),( Sung Won Jung ),( Jae Hyun Han ),( Joo Young Kim ) 대한간학회 2016 춘·추계 학술대회 (KASL) Vol.2016 No.1

Aims: There is widely known that numerous genomic variance involved in carcinogenesis. Recent advances in technology make it possible to approach to them closer. The aim of this study is to elicit critical genomic variance in different liver cancer. Methods: We sequenced short-insert (150bp, on average) genomic libraries of two primary multicentric liver tumors and one non-cancerous liver (NCL) tissue surgically resected from a male with chronic hepatitis B. The HiSeq X-Ten sequencer was used with 150- paired-end reads. Among two liver tumors, one was combined hepatocellular- cholangiocarcinoma (CHCCC) and the other was well differentiated hepatocellular carcinoma (HCC). After alignment to human reference genome and removal of duplications, three genomes were compared with each other. Results: We obtained nucleotide sequences covering 106.0 Gb CHCCC genome (37.1 x coverage), 102.6 Gb of HCC genome (35.9 x coverage), and 106.5 Gb (37.3 x coverage) of NCL genome. The sequenced reads covered 99.5% on all three genomes. Comparison of the CHCCC and NCL genomes showed 13,544 somatic single nucleotide variants (SNV), 3,789 small insertions and deletions, and 57 structural variants in CHCCC genome. Distinct SNVs were composed of 2.2% on exon, 37.3% on intron, and 60.5% on intergenic regions. Comparison of the HCC and NCL genomes showed 3,675 somatic single nucleotide variants (SNV), 3,491 small insertions and deletions, and 18 structural variants in HCC genome. Distinct SNVs in HCC were composed of 2.6% on exon, 39.9% on intron, and 57.5% on intergenic regions. Conclusions: The prevalence of somatic SNVs and structural variants in CHCCC is much more than HCC when compared with NCL. And that indicates that more complex process involved in CHCCC. Further researches will be performed for finding significant changes and process, and validation.

Integrated genome sizing (IGS) approach for the parallelization of whole genome analysis

Sona, Peter,Hong, Jong Hui,Lee, Sunho,Kim, Byong Joon,Hong, Woon-Young,Jung, Jongcheol,Kim, Han-Na,Kim, Hyung-Lae,Christopher, David,Herviou, Laurent,Im, Young Hwan,Lee, Kwee-Yum,Kim, Tae Soon,Jung, J BioMed Central 2018 BMC bioinformatics Vol.19 No.1

<P><B>Background</B></P><P>The use of whole genome sequence has increased recently with rapid progression of next-generation sequencing (NGS) technologies. However, storing raw sequence reads to perform large-scale genome analysis pose hardware challenges. Despite advancement in genome analytic platforms, efficient approaches remain relevant especially as applied to the human genome. In this study, an Integrated Genome Sizing (IGS) approach is adopted to speed up multiple whole genome analysis in high-performance computing (HPC) environment. The approach splits a genome (GRCh37) into 630 chunks (fragments) wherein multiple chunks can simultaneously be parallelized for sequence analyses across cohorts.</P><P><B>Results</B></P><P>IGS was integrated on Maha-Fs (HPC) system, to provide the parallelization required to analyze 2504 whole genomes. Using a single reference pilot genome, NA12878, we compared the NGS process time between Maha-Fs (NFS SATA hard disk drive) and SGI-UV300 (solid state drive memory). It was observed that SGI-UV300 was faster, having 32.5 mins of process time, while that of the Maha-Fs was 55.2 mins.</P><P><B>Conclusions</B></P><P>The implementation of IGS can leverage the ability of HPC systems to analyze multiple genomes simultaneously. We believe this approach will accelerate research advancement in personalized genomic medicine. Our method is comparable to the fastest methods for sequence alignment.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (10.1186/s12859-018-2499-1) contains supplementary material, which is available to authorized users.</P>

Comparative whole genome analysis of face-derived Streptococcus infantis CX-4 unravels the functions related to skin barrier

Lee Haeun,Lee Dong-Geol,Jo HyungWoo,Heo Young Mok,Baek Chaeyun,Kim Hye-Been,COSMAX BTI,Kang Seunghyun,Lee Wooseok,Mun Seyoung,Han Kyudong 한국유전학회 2024 Genes & Genomics Vol.46 No.4

Background The skin microbiome is essential in guarding against harmful pathogens and responding to environmental changes by generating substances useful in the cosmetic and pharmaceutical industries. Among these microorganisms, Streptococcus is a bacterial species identified in various isolation sources. In 2021, a strain of Streptococcus infantis, CX-4, was identified from facial skin and found to be linked to skin structure and elasticity. As the skin-derived strain differs from other S. infantis strains, which are usually of oral origin, it emphasizes the significance of bacterial variation by the environment. Objective This study aims to explore the unique characteristics of the CX-4 compared to seven oral-derived Streptococcus strains based on the Whole-Genome Sequencing data, focusing on its potential role in skin health and its possible application in cosmetic strategies. Methods The genome of the CX-4 strain was constructed using PacBio Sequencing, with the assembly performed using the SMRT protocol. Comparative whole-genome analysis was then performed with seven closely related strains, utilizing web-based tools like PATRIC, OrthoVenn3, and EggNOG-mapper, for various analyses, including protein association analysis using STRING. Results Our analysis unveiled a substantial number of Clusters of Orthologous Groups in diverse functional categories in CX-4, among which sphingosine kinase (SphK) emerged as a unique product, exclusively present in the CX-4 strain. SphK is a critical enzyme in the sphingolipid metabolic pathway, generating sphingosine-1-phosphate. The study also brought potential associations with isoprene formation and retinoic acid synthesis, the latter being a metabolite of vitamin A, renowned for its crucial function in promoting skin cell growth, differentiation, and maintaining of skin barrier integrity. These findings collectively suggest the potential of the CX-4 strain in enhancing of skin barrier functionality. Conclusion Our research underscores the potential of the skin-derived S. infantis CX-4 strain by revealing unique bacterial compounds and their potential roles on human skin. Background The skin microbiome is essential in guarding against harmful pathogens and responding to environmental changes by generating substances useful in the cosmetic and pharmaceutical industries. Among these microorganisms, Streptococcus is a bacterial species identified in various isolation sources. In 2021, a strain of Streptococcus infantis, CX-4, was identified from facial skin and found to be linked to skin structure and elasticity. As the skin-derived strain differs from other S. infantis strains, which are usually of oral origin, it emphasizes the significance of bacterial variation by the environment. Objective This study aims to explore the unique characteristics of the CX-4 compared to seven oral-derived Streptococcus strains based on the Whole-Genome Sequencing data, focusing on its potential role in skin health and its possible application in cosmetic strategies. Methods The genome of the CX-4 strain was constructed using PacBio Sequencing, with the assembly performed using the SMRT protocol. Comparative whole-genome analysis was then performed with seven closely related strains, utilizing web-based tools like PATRIC, OrthoVenn3, and EggNOG-mapper, for various analyses, including protein association analysis using STRING. Results Our analysis unveiled a substantial number of Clusters of Orthologous Groups in diverse functional categories in CX-4, among which sphingosine kinase (SphK) emerged as a unique product, exclusively present in the CX-4 strain. SphK is a critical enzyme in the sphingolipid metabolic pathway, generating sphingosine-1-phosphate. The study also brought potential associations with isoprene formation and retinoic acid synthesis, the latter being a metabolite of vitamin A, renowned for its crucial function in promoting skin cell growth, differentiation, and maintaining of skin barrier integrity. These findings collectively suggest the potential of the CX-4 strain in enhancing of skin barrier functionality. Conclusion Our research underscores the potential of the skin-derived S. infantis CX-4 strain by revealing unique bacterial compounds and their potential roles on human skin.

Whole genome sequencing of foot-and-mouth disease virus using benchtop next generation sequencing (NGS) system

Moon, Sung-Hyun,Oh, Yeonsu,Tark, Dongseob,Cho, Ho-Seong The Korean Society of Veterinary Service 2019 韓國家畜衛生學會誌 Vol.42 No.4

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.

Oxford Nanopore MinION을 이용한 SARS-CoV-2 유전체 분석 1예

김재석,정성희,김정민,김현수,김한성,송원근 대한임상미생물학회 2021 Annals of clinical microbiology Vol.24 No.4

The application of whole genome sequencing on SARS-CoV-2 viral genome is essential for ourunderstanding of the molecular epidemiology and spread of viruses in the community. Theportable whole genome sequencer MinION (Oxford Nanopore Technologies, ONT, UK) couldbe feasibly used in a clinical microbiology laboratory without the need of vast resources orstringent operating conditions. We used the MinION sequencer to analyze the viral genomesequence of one SARS-CoV-2 strain. In June 2020, nasopharyngeal specimen from one patientwas subjected to whole-genome analysis using the nCoV-2019 sequencing protocol v2 ofARTIC using the MinION sequencer. The ONT MinKNOW software, RAMPART tool, and GenomeWorkbench were used. We identified 11 nucleotide variants using the Wuhan-Hu-1 isolate(NC_045512.2) as the reference sequence. There were six nucleotide variants (T265I, F924,Y3884L, P4715L, L5462, and Q6804L) in the ORF1ab region, one variant (D614G) in the S gene,one variant (Q57H) in ORF3a, one variant (P302) in the N gene, and two variants in each the 5′-UTR and 3′-UTR. In this prolonged coronavirus disease 2019 (COVID-19) pandemic season, theMinION system that operates an amplicon-based whole-genome sequencing protocol could bea rapid and reliable sequencer without the need of cumbersome viral cultivation.