RESUMO
Synthetic oligonucleotides have become a fundamental tool in a wide range of biological fields, including synthetic biology, biosensing, and DNA storage. Reliable access to equipment for synthesizing high-density oligonucleotides in the laboratory ensures research security and the freedom of research expansion. In this study, we introduced the Open-Source Inkjet DNA Synthesizer (OpenIDS), an open-source inkjet-based microarray synthesizer that offers ease of construction, rapid deployment, and flexible scalability. Utilizing 3D printing, Arduino, and Raspberry Pi, this newly designed synthesizer achieved robust stability with an industrial inkjet printhead. OpenIDS maintains low production costs and is therefore suitable for self-fabrication and optimization in academic laboratories. Moreover, even non-experts can create and control the synthesizer with a high degree of freedom for structural modifications. Users can easily add printheads or alter the design of the microarray substrate according to their research needs. To validate its performance, we synthesized oligonucleotides on 144 spots on a 15 × 25-mm silicon wafer filled with controlled pore glass. The synthesized oligonucleotides were analyzed using urea polyacrylamide gel electrophoresis.
Assuntos
DNA , Oligonucleotídeos , DNA/química , Análise em MicrossériesRESUMO
The major drawbacks of RNA sequencing (RNA-seq), a remarkably accurate transcriptome profiling method, is its high cost and poor scalability. Here, we report a highly scalable and cost-effective method for transcriptomics profiling called Bulk transcriptOme profiling of cell Lysate in a single poT (BOLT-seq), which is performed using unpurified bulk 3'-end mRNA in crude cell lysates. During BOLT-seq, RNA/DNA hybrids are directly subjected to tagmentation, and second-strand cDNA synthesis and RNA purification are omitted, allowing libraries to be constructed in 2 h of hands-on time. BOLT-seq was successfully used to cluster small molecule drugs based on their mechanisms of action and intended targets. BOLT-seq competes effectively with alternative library construction and transcriptome profiling methods.
Assuntos
Perfilação da Expressão Gênica , RNA , RNA/genética , RNA Mensageiro/genética , Biblioteca Gênica , DNA Complementar/genética , Perfilação da Expressão Gênica/métodosRESUMO
PURPOSE: There have been needs to improve the sensitivity of liquid biopsy. This report aims to report the analytical and clinical validation of a next-generation sequencing (NGS)-based circulating tumor DNA (ctDNA) assay. MATERIALS AND METHODS: Analytical validation was conducted in vitro by evaluating the limit of detection (LOD), precision, and specificity for various genomic aberrations. The real-world performance in non-small cell lung cancer (NSCLC) was assessed by comparing the results of AlphaLiquid100 to the tissue-based results. RESULTS: The LODs with 30 ng input DNA were 0.11%, 0.11%, 0.06%, 0.21%, and 2.13 copies for detecting single nucleotide variants, insertions, deletions, fusions, and copy number alterations (CNA), respectively. Quantitatively, single nucleotide variants/insertions and deletions, fusions, and CNAs showed a good correlation (R2=0.91, 0.40, and 0.65; y=0.95, 1.06, and 1.19) to the manufacturer's values, and per-base specificities for all types of variants were near 100%. In real-world NSCLC (n=122), key actionable mutations in NSCLC were detected in 60.7% (74/122) with the ctDNA assay. Comparative analysis against the NGS-based tissue results for all key mutations showed positive percent agreement (PPA) of 85.3%. For individual genes, the PPA was as high as 95.7% for epidermal growth factor receptor (EGFR) mutations and 83.3% for ALK translocations. AlphaLiquid100 detected drug-sensitive EGFR mutation at a variant allele frequency as low as 0.02% and also identified an EGFR mutation in a case where tissue sample missed. Blood samples collected post-targeted therapies revealed additional acquired mutations. CONCLUSION: The AlphaLiquid100 ctDNA assay demonstrates robust analytical validity, offering clinically important information for NSCLC patients.
Assuntos
Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas , DNA Tumoral Circulante , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/sangue , DNA Tumoral Circulante/genética , DNA Tumoral Circulante/sangue , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/sangue , Mutação , Biópsia Líquida/métodos , Variações do Número de Cópias de DNA , Masculino , Feminino , Limite de Detecção , Sensibilidade e Especificidade , Pessoa de Meia-IdadeRESUMO
Cell-free DNA (cfDNA) sequencing has demonstrated great potential for early cancer detection. However, most large-scale studies have focused only on either targeted methylation sites or whole-genome sequencing, limiting comprehensive analysis that integrates both epigenetic and genetic signatures. In this study, we present a platform that enables simultaneous analysis of whole-genome methylation, copy number, and fragmentomic patterns of cfDNA in a single assay. Using a total of 950 plasma (361 healthy and 589 cancer) and 240 tissue samples, we demonstrate that a multifeature cancer signature ensemble (CSE) classifier integrating all features outperforms single-feature classifiers. At 95.2% specificity, the cancer detection sensitivity with methylation, copy number, and fragmentomic models was 77.2%, 61.4%, and 60.5%, respectively, but sensitivity was significantly increased to 88.9% with the CSE classifier (p value < 0.0001). For tissue of origin, the CSE classifier enhanced the accuracy beyond the methylation classifier, from 74.3% to 76.4%. Overall, this work proves the utility of a signature ensemble integrating epigenetic and genetic information for accurate cancer detection.
Assuntos
Ácidos Nucleicos Livres , Neoplasias , Humanos , Detecção Precoce de Câncer , Variações do Número de Cópias de DNA , Neoplasias/diagnóstico , Neoplasias/genética , Metilação de DNA , Biomarcadores Tumorais/genéticaRESUMO
Gynecologic cancer, including ovarian cancer and endometrial cancer, is characterized by morphological and molecular heterogeneity. Germline and somatic testing are available for patients to screen for pathogenic variants in genes such as BRCA1/2. Tissue expression levels of immunogenomic markers such as PD-L1 are also being used in clinical research. The basic therapeutic approach to gynecologic cancer combines surgery with chemotherapy. Immunotherapy, while not yet a mainstream treatment for gynecologic cancers, is advancing, with Dostarlimab recently receiving approval as a treatment for endometrial cancer. The goal remains to harness stimulated immune cells in the bloodstream to eradicate multiple metastases, a feat currently deemed challenging in a typical clinical setting. For the discovery of novel immunotherapy-based tumor targets, tumor-infiltrating lymphocytes (TILs) give a key insight on tumor-related immune activities by providing T cell receptor (TCR) sequences. Understanding the TCR repertoires of TILs in metastatic tissues and the circulation is important from an immunotherapy standpoint, as a subset of T cells in the blood have the potential to help kill tumor cells. To explore the relationship between distant tissue biopsy regions and blood circulation, we investigated the TCR beta chain (TCRß) in bulk tumor and matched blood samples from 39 patients with gynecologic cancer. We found that the TCR clones of TILs at different tumor sites were globally shared within patients and had high overlap with the TCR clones in peripheral blood.
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Neoplasias do Endométrio , Neoplasias Ovarianas , Humanos , Feminino , Proteína BRCA1 , Linfócitos do Interstício Tumoral , Proteína BRCA2 , Receptores de Antígenos de Linfócitos T/genética , Neoplasias Ovarianas/genética , Neoplasias do Endométrio/genéticaRESUMO
BACKGROUND: Postoperative minimal residual disease (MRD) detection using circulating-tumour DNA (ctDNA) requires a highly sensitive analysis platform. We have developed a tumour-informed, hybrid-capture ctDNA sequencing MRD assay. METHODS: Personalised target-capture panels for ctDNA detection were designed using individual variants identified in tumour whole-exome sequencing of each patient. MRD status was determined using ultra-high-depth sequencing data of plasma cell-free DNA. The MRD positivity and its association with clinical outcome were analysed in Stage II or III colorectal cancer (CRC). RESULTS: In 98 CRC patients, personalised panels for ctDNA sequencing were built from tumour data, including a median of 185 variants per patient. In silico simulation showed that increasing the number of target variants increases MRD detection sensitivity in low fractions (<0.01%). At postoperative 3-week, 21.4% of patients were positive for MRD by ctDNA. Postoperative positive MRD was strongly associated with poor disease-free survival (DFS) (adjusted hazard ratio 8.40, 95% confidence interval 3.49-20.2). Patients with a negative conversion of MRD after adjuvant therapy showed significantly better DFS (P < 0.001). CONCLUSION: Tumour-informed, hybrid-capture-based ctDNA assay monitoring a large number of patient-specific mutations is a sensitive strategy for MRD detection to predict recurrence in CRC.
Assuntos
DNA Tumoral Circulante , Neoplasias Colorretais , Humanos , DNA Tumoral Circulante/genética , Neoplasia Residual/genética , Intervalo Livre de Doença , Mutação , Biomarcadores Tumorais/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genéticaRESUMO
Traditional tissue-based assessments of genomic alterations in castration-resistant prostate cancer (CRPC) can be challenging. To evaluate the real-world clinical utility of liquid biopsies for the evaluation of genomic alterations in CRPC, we preemptively collected available plasma samples and archival tissue samples from patients that were being treated for clinically confirmed CRPC. The cell-free DNA (cfDNA) and tumor tissue DNA were analyzed using the AlphaLiquid®100-HRR panel. Plasma samples from a total of 87 patients were included in this study. Somatic mutations from cfDNA were detected in 78 (89.7%) patients, regardless of the presence of overt metastasis or concomitant treatment given at the time of plasma sample collection. Twenty-three patients were found to have known deleterious somatic or germline mutations in HRR genes from their cfDNA. Archival tissue samples from 33 (37.9%) patients were available for comparative analysis. Tissue sequencing was able to yield an NGS result in only 51.5% of the tissue samples. The general sensitivity of cfDNA for detecting somatic mutations in tissues was 71.8%, but important somatic/germline mutations in HRR genes were found to have a higher concordance (100%). Liquid biopsies can be a reasonable substitute for tissue biopsies in CRPC patients when evaluating genomic alterations.
RESUMO
Patients with hematuria are commonly given an invasive cystoscopy test to detect bladder cancer (BC). To avoid the risks associated with cystoscopy, several urine-based methods for BC detection have been developed, the most prominent of which is the deep sequencing of urine DNA. However, the current methods for urine-based BC detection have significant levels of false-positive signals. In this study, we report on uAL100, a method to precisely detect BC tumor DNA in the urine without tumor samples. Using urine samples from 43 patients with BC and 21 healthy donors, uAL100 detected BC with 83.7% sensitivity and 100% specificity. The mutations identified in the urine DNA by uAL100 for BC detection were highly associated with BC tumorigenesis and progression. We suggest that uAL100 has improved accuracy compared to other urine-based methods for early BC detection and can reduce unnecessary cystoscopy tests for patients with hematuria.
RESUMO
BACKGROUND: A growing body of research has emphasized 5-hydroxymethylcytosine (5hmC) as an important epigenetic mark. High-resolution methods to detect 5hmC require high sequencing depth and are therefore expensive. Many studies have used enrichment-based methods to detect 5hmC; however, conventional enrichment-based methods have limited resolution. To overcome these limitations, we developed EBS-seq, a cost-efficient method for 5hmC detection with single-base resolution that combines the advantages of high-resolution methods and enrichment-based methods. RESULTS: EBS-seq uses selective labeling of 5hmC, deamination of cytosine and 5-methylcytosine, pull-down of labeled 5hmC, and C-to-T conversion during DNA amplification. Using this method, we profiled 5hmC in HEK293T cells and two colorectal cancer samples. Compared with conventional enrichment-based 5hmC detection, EBS-seq improved 5hmC signals by localizing them at single-base resolution. Furthermore, EBS-seq was able to determine 5hmC levels in CpG-dense regions where distortion of signals can occur, such as CpG islands and CpG shores. Comparing EBS-seq and conventional high-resolution 5hmC detection by ACE-seq, we showed that EBS-seq is more effective at finding 5hmC sites. Using EBS-seq, we found strong associations between gene expression and gene-body 5hmC content in both HEK293T cells and colorectal cancer samples. CONCLUSIONS: EBS-seq is a reliable and cost-efficient method for 5hmC detection because it simultaneously enriches 5hmC-containing DNA fragments and localizes 5hmC signals at single-base resolution. This method is a promising choice for 5hmC detection in challenging clinical samples with low 5hmC levels, such as cancer tissues.
Assuntos
5-Metilcitosina , Neoplasias Colorretais , Humanos , Células HEK293 , Metilação de DNARESUMO
Poly(ADP-ribose) polymerase inhibitors have been shown dramatic responses in patients with BRCAness. However, clinical studies have been limited to breast cancer patients with germline mutations. Here, we describe a patient with metastatic breast cancer who had a rare BRCA1 somatic mutation (BRCA1 c.4336G>T (p.E1446*)) detected by cell-free DNA analysis after failing standard therapies. This tier III variant of unknown significance was predicted to be a pathogenic variant in our assessment, leading us to consider off-label treatment with olaparib. The patient responded well to olaparib for several months, with a decrease in allele frequency of this BRCA1 somatic mutation in cell-free DNA. Olaparib resistance subsequently developed with an increase in the allele frequency and new BRCA1 reversion mutations. To our knowledge, this is the first report confirming BRCA1 c.4336G>T (p.E1446*) as a mutation sensitive to olaparib in breast cancer and describing the dynamic changes in the associated mutations using liquid biopsy.
Assuntos
Neoplasias da Mama , Ácidos Nucleicos Livres , DNA Tumoral Circulante , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , DNA Tumoral Circulante/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Mutação , Ácidos Nucleicos Livres/uso terapêuticoRESUMO
BACKGROUND: The factors affecting cardioprotective collateral circulation are still incompletely understood. Recently, characteristics, such as CpG methylation of cell-free DNA (cfDNA), have been reported as markers with clinical utility. The aim of this study was to evaluate whether cfDNA methylation patterns are associated with the grade of coronary collateral circulation (CCC). RESULT: In this case-control study, clinical and angiographic data were obtained from 143 patients (mean age, 58 years, male 71%) with chronic total coronary occlusion. Enzymatic methyl-sequencing (EM-seq) libraries were prepared using the cfDNA extracted from the plasma. Data were processed to obtain the average methylation fraction (AMF) tables of genomic regions from which blacklisted regions were removed. Unsupervised analysis of the obtained AMF values showed that some of the changes in methylation were due to CCC. Through random forest preparation process, 256 differentially methylated region (DMR) candidates showing strong association with CCC were selected. A random forest classifier was then constructed, and the area under the curve of the receiver operating characteristic curve indicated an appropriate predictive function for CCC. Finally, 20 DMRs were identified to have significantly different AMF values between the good and poor CCC groups. Particularly, the good CCC group exhibited hypomethylated DMRs. Pathway analysis revealed five pathways, including TGF-beta signaling, to be associated with good CCC. CONCLUSION: These data have demonstrated that differential hypomethylation was identified in dozens of cfDNA regions in patients with good CCC. Our results support the clinical utility of noninvasively obtained epigenetic signatures for predicting collateral circulation in patients with vascular diseases.
Assuntos
Ácidos Nucleicos Livres , Doença da Artéria Coronariana , Humanos , Masculino , Pessoa de Meia-Idade , Estudos de Casos e Controles , Circulação Colateral , Metilação de DNA , FemininoRESUMO
The development of single-cell RNA-seq has broadened the spectrum for biological research by providing a high-resolution analysis of cellular heterogeneity. However, the requirement for sophisticated devices for the compartmentalization of cells has limited its widespread applicability. Here, we develop Onepot-Seq, a device-free method, that harnesses the transient localization of mRNA after lysis to capture single-cell transcriptomes simultaneously in a continuous fluid medium. In mixed-species experiments, we obtained high-quality single-cell profiles. Further, cell type-specific poly(A)-conjugated antibodies allow Onepot-Seq to effectively capture target cells in complex populations. Chemical perturbations to cells can be profiled by Onepot-Seq at single-cell resolution. Onepot-Seq should allow routine transcriptional profiling at single-cell resolution, accelerating clinical and scientific discoveries in many fields of science.
This article describes a strategy for single-cell RNA sequencing that avoids the usual imitation required by first physically compartmentalizing single cells. Based on the slow rate of mRNA diffusion relative to mRNA capture on beads, the authors devised a method that profiles single-cell mRNA among a multiple cell population, within one reaction mixture, termed 'Onepot-Seq'. This approach uses a time-constrained, transient reaction chamber that forms around each cell, with only slow diffusion of the cell contents after gentle lysis. The sparse distribution of cells and beads allows the beads to capture the profile of a single cell with minimal intercellular mRNA contamination.
Assuntos
Análise de Célula Única , Transcriptoma , Transcriptoma/genética , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Perfilação da Expressão Gênica/métodosRESUMO
BACKGROUND: Circulating tumour DNA (ctDNA) has been spotlighted as an attractive biomarker because of its easy accessibility and real-time representation of tumour genetic profile. However, the clinical utility of longitudinal ctDNA monitoring has not been clearly defined. METHODS: Serial blood samples were obtained from metastatic colorectal cancer patients undergoing first-line chemotherapy. ctDNA was sequenced using a targeted next-generation sequencing platform which included 106 genes. Changes in ctDNA profile and treatment outcome were comprehensively analysed. RESULTS: A total of 272 samples from 62 patients were analysed. In all, 90.3% of patients had detectable ctDNA mutation before treatment. ctDNA clearance after chemotherapy was associated with longer progression-free survival which was independent of radiological response (adjusted hazard ratio 0.22, 95% confidence interval 0.10-0.46). Longitudinal monitoring was able to detect ctDNA progression which preceded radiological progressive disease (PD) in 58.1% (median 3.3 months). Diverse resistant mutations (34.9%) and gene amplification (7.0%) at the time of PD were discovered. For 16.3% of the PD patients, the newly identified mutations could be potential candidates of targeted therapy or clinical trial. CONCLUSION: ctDNA profile provided a more accurate landscape of tumour and dynamic changes compared to radiological evaluation. Longitudinal ctDNA monitoring may improve personalised treatment decision-making.
Assuntos
DNA Tumoral Circulante , Neoplasias do Colo , Neoplasias Colorretais , Neoplasias Retais , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , DNA de Neoplasias/genética , Humanos , MutaçãoRESUMO
Neoantigen vaccines are an immunotherapy strategy for treating cancer. The vaccine degrades quickly, so the strategy must include protection and precise targeting for immune cell stimulation. In this study, we engineered attenuated Salmonella typhimurium, which is highly infiltrative to tumors, to act as a carrier for Neoantigen peptide vaccine. Our system used a constitutive promoter vector, so that a single injection of Salmonella expressing Neoantigen could be used without requiring additional induction injections. In vivo experiments on bacteria-treated mice showed that Neoantigen expressed by the engineered carrier infiltrated tumors and resulted in suppressed tumor growth, higher survival rates and longer survival times, a relative increase of CD4 and CD8 T cells, and cytokine release. These results indicate that engineered Salmonella can be used as a carrier for Neoantigen immunotherapy.
Assuntos
Antígenos/uso terapêutico , Engenharia Genética , Imunoterapia/métodos , Neoplasias Experimentais/terapia , Salmonella typhimurium/imunologia , Animais , Antígenos/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Salmonella typhimurium/genética , Taxa de Sobrevida , Microambiente TumoralRESUMO
DNA methylation is an epigenetic mechanism that is related to mammalian cellular differentiation, gene expression regulation, and disease. In several studies, DNA methylation has been identified as an effective marker to identify differences between cells. In this review, we introduce single-cell DNA-methylation profiling methods, including experimental strategies and approaches to computational data analysis. Furthermore, the blind spots of the basic analysis and recent alternatives are briefly described. In addition, we introduce well-known applications and discuss future development.
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Metilação de DNA , Perfilação da Expressão Gênica , Análise de Célula Única , Animais , HumanosRESUMO
We aimed to identify somatic genetic alterations in pure growth hormone (GH)-secreting pituitary adenomas without GNAS variants. Patients with GH-secreting pituitary adenoma who underwent transsphenoidal adenomectomy at Severance Hospital, Yonsei University College of Medicine were recruited. Somatic genetic alterations were profiled by whole-exome sequencing (WES) and targeted resequencing. WES was performed using DNA from nine GH-secreting pituitary tumors and corresponding blood samples. Absence of GNAS variant was confirmed by Sanger sequencing. For targeted resequencing of 140 fixed tissues, 48 WES-derived candidate genes and 7 GH-secreting pituitary adenoma-associated genes were included. Forty-eight genes with 59 somatic variants were identified by WES. In targeted resequencing, variants in 26 recurrent genes, including MAST4, PRIM2, TNN, STARD9, DNAH11, DOCK4, GPR98, BCHE, DARS, CUBN, NGDN, PLXND1, UNC5B, and COL22A1, were identified, but variants in previously reported genes were not detected. BCHE, DARS, NGDN, and UNC5B variants were associated with increased GH-secreting pituitary tumor biochemical activity, which was confirmed in vitro. Although recurrent point variants were rare, several somatic variants were identified in sporadic pure GH-secreting pituitary adenomas. Several somatic variants may affect pathways involved in the tumorigenesis and biochemical activities of GH-secreting pituitary adenomas.
Assuntos
Adenoma/genética , Genes Neoplásicos , Adenoma Hipofisário Secretor de Hormônio do Crescimento/genética , Neoplasias Hipofisárias/genética , Adenoma/metabolismo , Adulto , Idoso , Animais , Biomarcadores Tumorais/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Cromograninas , DNA de Neoplasias/genética , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Ontologia Genética , Estudos de Associação Genética , Adenoma Hipofisário Secretor de Hormônio do Crescimento/metabolismo , Hormônio do Crescimento Humano/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Hipofisárias/metabolismo , Ratos , Análise de Sequência de DNA , Transfecção , Sequenciamento do Exoma , Adulto JovemRESUMO
The detection of low-frequency somatic mutations enables early diagnosis of disease; however, base-substitution errors that arise during genomic library preparation and high-throughput sequencing can lead to false diagnostic information. To discriminate true genomic alterations from technical errors, we developed spCas9-assisted true variant labeling sequencing (CARVE-seq), which detects low-frequency mutant alleles with high accuracy. CARVE-seq utilizes single-base discrimination during spCas9 cleavage reactions to exclude technical errors. Ten single nucleotide variants that recurrently occur in tumors were assayed by CARVE-seq using 20 ng reference samples, and 100% positive predictive value and specificity was observed, which proved the highly accurate performance of CARVE-seq.
Assuntos
Alelos , Sistemas CRISPR-Cas , Confiabilidade dos Dados , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Proteína 9 Associada à CRISPR/genética , Edição de Genes/métodos , Humanos , Neoplasias/sangue , Valor Preditivo dos Testes , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Recently developed single-cell RNA sequencing methods allow the simultaneous profiling of the transcriptomes of thousands of individual cells. However, current methods still require advanced equipment or entail substantial waste of reagents. Here, we introduce magnetic bead-assisted parallel single-cell gene expression sequencing (MAPS-seq), a microwell-based method that pools samples before the reverse transcription step, increasing the ease of sample preparation and reducing reagent waste. Moreover, because this method uses universal reagents and standard molecular biology lab instruments, it is easy to implement, even in labs that have not previously conducted single-cell RNA sequencing. We validated our method by demonstrating that it can generate gene expression data at the single-cell level. We then applied the MAPS-seq method to analyze 237 human myelogenous leukemia cells treated with one of three different drugs or dimethyl sulfoxide. We observed transcriptional changes and identified marker genes that indicate a drug response. Furthermore, the MAPS-seq method produced data of comparable quality to those of existing single-cell RNA sequencing methods. Consequently, we expect that our method will provide researchers with a more accessible, less wasteful, and less burdensome method for investigating the transcriptomes of individual cells.
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Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nanopartículas Magnéticas de Óxido de Ferro , Análise de Célula Única/métodos , Transcriptoma , Linhagem Celular , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Reprodutibilidade dos Testes , Análise de Sequência de RNA/métodosRESUMO
Analyzing cell-free DNA (cfDNA) as a source of circulating tumor DNA is useful for diagnosing or monitoring patients with cancer. However, the concordance between cfDNA within liquid biopsy and genomic DNA (gDNA) within tumor tissue biopsy is still under debate. To evaluate the concordance in a clinical setting, we enrolled 54 patients with metastatic colorectal cancer and analyzed their plasma cfDNA, gDNA from peripheral blood mononuclear cells (PBMC), and gDNA from available matched tumor tissues using ultra-deep sequencing targeting 10 genes (38-kb size) recurrently mutated in colorectal cancer. We first established a highly reliable cut-off value using reference material. The sensitivity of detecting KRAS hotspot mutations in plasma was calculated as 100%, according to digital droplet PCR. We could selectively detect clinically important somatic alterations with a variant allele frequency as low as 0.18%. We next compared somatic mutations of the 10 genes between cfDNA and genomic DNA from tumor tissues and observed an overall 93% concordance rate between the two types of samples. Additionally, the concordance rate of patients with the time interval between liquid biopsy and tumor tissue biopsy within 6 months and no prior exposure to chemotherapy was much higher than those without. The patients with KRAS mutant fragments in plasma had poor prognosis than those without the mutant fragments (33 months vs. 63 months; p<0.05). Consequently, the profiling with our method could achieve highly concordant results and may facilitate the surveillance of the tumor status with liquid biopsy in CRC patients.