• Title, Summary, Keyword: single-cell genomics

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Single-Cell Sequencing in Cancer: Recent Applications to Immunogenomics and Multi-omics Tools

  • Sierant, Michael C.;Choi, Jungmin
    • Genomics & Informatics
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    • v.16 no.4
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    • pp.17.1-17.6
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    • 2018
  • Tumor heterogeneity, the cellular mosaic of multiple lineages arising from the process of clonal evolution, has continued to thwart multi-omics analyses using traditional bulk sequencing methods. The application of single-cell sequencing, in concert with existing genomics methods, has enabled high-resolution interrogation of the genome, transcriptome, epigenome, and proteome. Applied to cancers, these single-cell multi-omics methods bypass previous limitations on data resolution and have enabled a more nuanced understanding of the evolutionary dynamics of tumor progression, immune evasion, metastasis, and treatment resistance. This review details the growing number of novel single-cell multi-omics methods applied to tumors and further discusses recent discoveries emerging from these approaches, especially in regard to immunotherapy.

Identification of ERBB pathway-activated cells in triple-negative breast cancer

  • Cho, Soo Young
    • Genomics & Informatics
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    • v.17 no.1
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    • pp.3.1-3.4
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    • 2019
  • Intratumor heterogeneity within a single tumor mass is one of the hallmarks of malignancy and has been reported in various tumor types. The molecular characterization of intratumor heterogeneity in breast cancer is a significant challenge for effective treatment. Using single-cell RNA sequencing (RNA-seq) data from a public resource, an ERBB pathway activated triple-negative cell population was identified. The differential expression of three subtyping marker genes (ERBB2, ESR1, and PGR) was not changed in the bulk RNA-seq data, but the single-cell transcriptomes showed intratumor heterogeneity. This result shows that ERBB signaling is activated using an indirect route and that the molecular subtype is changed on a single-cell level. Our data propose a different view on breast cancer subtypes, clarifying much confusion in this field and contributing to precision medicine.

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.

Identification of Polymorphisms of Fas Gene and Association Analysis in Hanwoo

  • Kim, Seung-Chang;Lee, Seung-Hwan;Kim, Bum-Soo;Kim, Tae-Hun;Seong, Hwan-Hoo;Oh, Sung-Jong;Yoon, Du-Hak;Choi, Bong-Hwan
    • Journal of Animal Science and Technology
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    • v.53 no.6
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    • pp.511-516
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    • 2011
  • Fas gene known to associate with intramuscular fat content in Korean cattle was selected for DNA marker development. Fas (APO-1, CD95), a member of the tumor necrosis factor (TNF) receptor superfamily, is a cell membrane protein that mediates apoptosis (programmed cell death). We discovered single nucleotide polymorphisms (SNPs) within Fas gene in order to develop novel DNA markers at genomic level. Of this gene to search for SNP, sequences of whole exon and 1kb range of both front and back of the gene using 24 cattle were determined by direct-sequencing methods. As a result, 16 SNPs in exon, 37 SNPs in intron and 2 SNPs in promoter region, a total of 55 SNPs were discovered. In these SNPs, thirty-one common polymorphic sites were selected considering their allele frequencies, haplotype-tagging status and Linkage Disequilibrium (LD) for genotyping in larger-scale subjects. Selected SNPs were confirmed genotype through SNaPshot method (n=274) and were examined for possible genetic association of Fas polymorphisms with carcass weight (CWT), eye muscle area (EMA), and backfat thickness (BF). So, the SNP have been identified significant g.-12T>G, g.1112T>G and g.32548T>C. These results suggest that polymorphism of Fas gene was associated with meat quality traits in Hanwoo.

Single Nucleotide Polymorphism in the Promoter Region of H1 Histone Family Member N, Testis-specific (H1FNT) and Its Association Study with Male Infertility

  • Yang, Seung-Hee;Lee, Jin-U;Lee, Su-Man
    • Genomics & Informatics
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    • v.8 no.4
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    • pp.201-205
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    • 2010
  • The H1 histone family, member N, testis-specific (H1FNT) is exclusively expressed in the testis, and had its possible role for sperm chromatin formation. The purpose of this study is to investigate any genetic association of H1FNT gene with male infertility, especially at the promoter region. We examined the promoter single nucleotide polymorphisms (SNP) of H1FNT gene which is located within transcription factor binding site for its association with male infertility. The statistical analysis showed that the -1129A>T polymorphism was present at a statistically significance in male infertility (p=0.0059 and 0.0349 for hetero and risk type, respectively). The dual-luciferase promoter assay was performed to examine the polymorphic effect of this promoter SNP by the cloning of promoter region (1700bp fragment) into pGL3-basic vector. In our plasmid based reporter system, there is no big difference between wild and risk type. In conclusion, H1FNT -1129A>T promoter SNP is statistically significant with male infertility, especially with subfertile (non-azoospermia) group. Further analysis of its functional polymorphic effect in vivo may provide the biological significance of testis-specific histone with spermatogenesis.

Silencing of CaCDPK4 ( Capsicum annuum Calcium Dependent Protein Kinase) and ItsOrtholog, NbCDPK5 Induces Cell Death in Nicotiana benthamiana

  • Eunsook Chung;Kim, Young-Cheol;Oh, Sang-Keun;Younghee Jung;Kim, Soo-Yong;Park, Doil
    • Proceedings of the Korean Society of Plant Pathology Conference
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    • pp.77.1-77
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    • 2003
  • We have isolated a full-length cDNA clone, CaCDPK4 encoding a typical calcium-dependent protein kinase (CDPK) from hot pepper cDNA library. Genomic southern blot analysis showed that it belongs to a multigene family, but represents a single copy gone in hot pepper genome. RNA expression pattern of this gene revealed that it is induced by infiltration of Xanthomonas axonopodis pv. glycines Bra into hot pepper leaves but not by water deficit stress. However, high salt treatment of NaCl (0.4 M) solution to hot pepper plants strongly induced CaCDPK4 gene. In addition, this gene is weakly responsive to the exogenous application of salicylic acid or ethephon. Biochemical study of the GST-CaCDPK4 recominant protein showed that it autophosphorylates in vitro and the presence of EGTA, a calcium chelater, eliminates the kinase activity of the recombinant protein. As a way to identify the in vivo function of CaCDPK4 in plants, VIGS (Virus-Induced Gene Silencing) was employed. Agrobacterium-mediated TRV silencing construct containing the kinase and calmodulin domain of CaCDPK4 resulted in cell death of Nicotiana benthamiana plants. A highly homologous H benthamiana CDPK gene, NbCDPK5, to CaCDPK4 was cloned from N. benthamiana cDNA library. VIGS of NbCDPK5 also resulted in cell death. The molecular characterization of this cell death phenotype is being under investigation.

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Identification of Single Nucleotide Polymorphism Marker and Association Analysis of Marbling Score in Fas Gene of Hanwoo

  • Kim, Seung-Chang;Lee, Seung-Hwan;Lee, Ji-Woong;Kim, Tae-Hun;Choi, Bong-Hwan
    • Asian-Australasian Journal of Animal Sciences
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    • v.29 no.1
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    • pp.23-28
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    • 2016
  • The Fas (APO-1, TNFRSF6) gene known as a member of the tumor necrosis factor receptor superfamily was selected for DNA marker development in Korean cattle. It is a cell membrane protein and mediates programmed cell death (apoptosis). We discovered single nucleotide polymorphisms (SNPs) within Fas gene in order to develop novel DNA markers related to economical traits at the genomic level. The sequences of whole exon and 1 kb range of both front and back of the gene were determined by direct-sequencing methods using 24 cattle. A total of 55 SNPs were discovered and we selected 31 common polymorphic sites considering their allele frequencies, haplotype-tagging status and linkage disequilibrium (LD) for genotyping in larger-scale subjects. The SNPs were confirmed genotype through the SNaPshot method (n = 274) and were examined for a possible genetic association between Fas polymorphisms and marbling score. So, the SNPs that were identified significant are g.30256G>C, g.31474C>A, g.31940A>G, and g.32982G>A. These results suggest that SNPs of Fas gene were associated with intramuscular fat content of meat quality traits in Korean cattle.

Functional Genomics in the Context of Biocatalysis and Biodegradation

  • Koh Sung-Cheol;Kim Byung-Hyuk
    • Proceedings of the Microbiological Society of Korea Conference
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    • pp.3-14
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    • 2002
  • Functional genomics aims at uncovering useful information carried on genome sequences and at using it to understand the mechanisms of biological function. Elucidating the unknown biological functions of new genes based upon the genomics rationales will greatly speed up the extensive understanding of biocatalysis and biodegradation in biological world including microorganisms. DNA microarrays generate a system for the simultaneous measurement of the expression level of thousands of genes in a single hybridization assay. Their data mining (transcriptome) strategy has two categories: differential gene expression and coordinated gene expression. Furthermore, measurement of proteins (proteome) generates information on how the transcribed sequences end up as functional characteristics within the cell, and quantitation of metabolites yields information on how the functional proteins act to produce energy and process substrates (metabolome). Various composite functional genomics databases containing genetic, enzymatic and metabolic information have been developed and will contribute to the understanding of the life blue print and the new discoveries and practices in biocatalysis and biodegradation that could enrich their industrial and environmental applications.

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Rice 7-Hydroxymethyl Chlorophyll a Reductase Is Involved in the Promotion of Chlorophyll Degradation and Modulates Cell Death Signaling

  • Piao, Weilan;Han, Su-Hyun;Sakuraba, Yasuhito;Paek, Nam-Chon
    • Molecules and Cells
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    • v.40 no.10
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    • pp.773-786
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    • 2017
  • The loss of green coloration via chlorophyll (Chl) degradation typically occurs during leaf senescence. To date, many Chl catabolic enzymes have been identified and shown to interact with light harvesting complex II to form a Chl degradation complex in senescing chloroplasts; this complex might metabolically channel phototoxic Chl catabolic intermediates to prevent oxidative damage to cells. The Chl catabolic enzyme 7-hydroxymethyl Chl a reductase (HCAR) converts 7-hydroxymethyl Chl a (7-HMC a) to Chl a. The rice (Oryza sativa) genome contains a single HCAR homolog (OsHCAR), but its exact role remains unknown. Here, we show that an oshcar knockout mutant exhibits persistent green leaves during both dark-induced and natural senescence, and accumulates 7-HMC a and pheophorbide a (Pheo a) in green leaf blades. Interestingly, both rice and Arabidopsis hcar mutants exhibit severe cell death at the vegetative stage; this cell death largely occurs in a light intensity-dependent manner. In addition, 7-HMC a treatment led to the generation of singlet oxygen ($^1O_2$) in Arabidopsis and rice protoplasts in the light. Under herbicide-induced oxidative stress conditions, leaf necrosis was more severe in hcar plants than in wild type, and HCAR-overexpressing plants were more tolerant to reactive oxygen species than wild type. Therefore, in addition to functioning in the conversion of 7-HMC a to Chl a in senescent leaves, HCAR may play a critical role in protecting plants from high light-induced damage by preventing the accumulation of 7-HMC a and Pheo a in developing and mature leaves at the vegetative stage.

Prostate Stem Cell Antigen Single Nucleotide Polymorphisms Influence Risk of Estrogen Receptor Negative Breast Cancer in Korean Females

  • Kim, Sook-Young;Yoo, Jae-Young;Shin, Ae-Sun;Kim, Yeon-Ju;Lee, Eun-Sook;Lee, Yeon-Su
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.1
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    • pp.41-48
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    • 2012
  • Introduction: Breast cancer is the second leading cancer in Korean women. To assess potential genetic associations between the prostate stem cell antigen (PSCA) gene in the chromosome 8q24 locus and breast cancer risk in Korean women, 13 SNPs were selected and associations with breast cancer risk were analyzed with reference to hormone receptor (HR) and menopausal status. Methods:We analyzed DNA extracted from buffy coat from 456 patients and 461 control samples, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) based upon region-specific PCR followed by allelespecific single base primer extension reactions. Risks associated with PSCA genotypes and haplotypes were estimated with chi-square test (${\chi}^2$-test), and polytomous logistic regression models using odds ratios (OR) and 95% confidence intervals (CIs), by HR and menopausal status. Results: In case-control analysis, odds ratios (OR) of rs2294009, rs2294008, rs2978981, rs2920298, rs2976395, and rs2976396 were statistically significant only among women with estrogen receptor (ER) negative cancers, and those of rs2294008, rs2978981, rs2294010, rs2920298, rs2976394, rs10216533, and rs2976396 were statistically significant only in pre-menopausal women, and not in postmenopausal women. Risk with the TTGGCAA haplotype was significantly elevated in ER (-) status (OR= 1.48, 95% CI= 1.03~2.12, p<0.05). Especially risk of allele T of rs2294008 is significantly low in pre-menopausal breast cancer patients and AA genotype of rs2976395 in ER (-) status represents the increase of OR value. Conclusion: This report indicated for the first time that associations exist between PSCA SNPs and breast cancer susceptibility in Korean women, particularly those who are pre-menopausal with an estrogen receptor negative tumor status.