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1.
Nucleic Acids Res ; 46(7): e40, 2018 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-29361040

RESUMO

Scalable and cost-effective production of error-free DNA is critical to meet the increased demand for such DNA in the field of biological science. Methods based on 'Dial-out PCR' have enabled the high-throughput error-free DNA synthesis from a microarray-synthesized DNA pool by labeling with retrieval PCR tags, and retrieving error-free DNA of which the sequence is identified via next generation sequencing (NGS). However, most of the retrieved products contain byproducts due to background amplification of redundantly labeled DNAs. Here, we present a highly selective retrieval method of desired DNA from a pool of millions of DNA clones from NGS platforms. Our strategy is based on replicating entire sequence-verified DNA molecules from NGS plates to obtain population-controlled DNA pool. Using the NGS-replica pool, we could perform improved and selective retrieval of desired DNA from the replicated DNA pool compared to other dial-out PCR based methods. To evaluate the method, we tested this strategy by using 454, Illumina, and Ion Torrent platforms for producing NGS-replica pool. As a result, we observed a highly selective retrieval yield of over 95%. We anticipate that applications based on this method will enable the preparation of high-fidelity sequenced DNA from heterogeneous collections of DNA molecules.


Assuntos
DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase/métodos , Replicação do DNA/genética , Humanos , Análise de Sequência de DNA/métodos
2.
BMC Cancer ; 19(1): 421, 2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-31060539

RESUMO

BACKGROUND: Although the prognostic biomarkers associated with colorectal cancer (CRC) survival are well known, there are limited data on the association between the molecular characteristics and survival after recurrence (SAR). The purpose of this study was to assess the association between pathway mutations and SAR. METHODS: Of the 516 patients with stage III or high risk stage II CRC patients treated with surgery and adjuvant chemotherapy, 87 who had distant recurrence were included in the present study. We analyzed the association between SAR and mutations of 40 genes included in the five critical pathways of CRC (WNT, P53, RTK-RAS, TGF-ß, and PI3K). RESULTS: Mutation of genes within the WNT, P53, RTK-RAS, TGF-ß, and PI3K pathways were shown in 69(79.3%), 60(69.0%), 57(65.5%), 21(24.1%), and 19(21.8%) patients, respectively. Patients with TGF-ß pathway mutation were younger and had higher incidence of mucinous adenocarcinoma (MAC) histology and microsatellite instability-high. TGF-ß pathway mutation (median SAR of 21.6 vs. 44.4 months, p = 0.021) and MAC (20.0 vs. 44.4 months, p = 0.003) were associated with poor SAR, and receiving curative resection after recurrence was associated with favorable SAR (Not reached vs. 23.6 months, p <  0.001). Mutations in genes within other critical pathways were not associated with SAR. When MAC was excluded as a covariate, multivariate analysis revealed TGF-ß pathway mutation and curative resection after distant recurrence as an independent prognostic factor for SAR. The impact of TGF-ß pathway mutations were predicted using the PROVEAN, SIFT, and PolyPhen-2. Among 25 mutations, 23(92.0%)-24(96.0%) mutations were predicted to be damaging mutation. CONCLUSIONS: Mutation in genes within TGF-ß pathway may have negative prognostic role for SAR in CRC. Other pathway mutations were not associated with SAR.


Assuntos
Adenocarcinoma Mucinoso/genética , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Recidiva Local de Neoplasia/genética , Transdução de Sinais/genética , Adenocarcinoma Mucinoso/mortalidade , Adenocarcinoma Mucinoso/patologia , Adenocarcinoma Mucinoso/terapia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/metabolismo , Capecitabina , Quimioterapia Adjuvante/métodos , Colo/patologia , Colo/cirurgia , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Feminino , Fluoruracila/análogos & derivados , Fluoruracila/uso terapêutico , Seguimentos , Humanos , Leucovorina/uso terapêutico , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/terapia , Compostos Organoplatínicos/uso terapêutico , Oxaloacetatos , Cuidados Paliativos/métodos , Prognóstico , Reto/patologia , Reto/cirurgia , Análise de Sobrevida , Fator de Crescimento Transformador beta/metabolismo
3.
Cancer ; 123(18): 3513-3523, 2017 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-28513830

RESUMO

BACKGROUND: Colorectal cancer (CRC) develops through the alteration of several critical pathways. This study was aimed at evaluating the influence of critical pathways on survival outcomes for patients with CRC. METHODS: Targeted next-generation sequencing of 40 genes included in the 5 critical pathways of CRC (WNT, P53, RTK-RAS, phosphatidylinositol-4,5-bisphosphate 3-kinase [PI3K], and transforming growth factor ß [TGF-ß]) was performed for 516 patients with stage III or high-risk stage II CRC treated with surgery followed by adjuvant fluoropyrimidine and oxaliplatin chemotherapy. The associations between critical pathway mutations and relapse-free survival (RFS) and overall survival were analyzed. The associations were further analyzed according to the tumor location. RESULTS: The mutation rates for the WNT, P53, RTK-RAS, PI3K, and TGF-ß pathways were 84.5%, 69.0%, 60.7%, 30.0%, and 28.9%, respectively. A mutation in the PI3K pathway was associated with longer RFS (adjusted hazard ratio [HR], 0.59; 95% confidence interval [CI], 0.36-0.99), whereas a mutation in the RTK-RAS pathway was associated with shorter RFS (adjusted HR, 1.60; 95% CI, 1.01-2.52). Proximal tumors showed a higher mutation rate than distal tumors, and the mutation profile was different according to the tumor location. The mutation rates of Kirsten rat sarcoma viral oncogene homolog (KRAS), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA), and B-Raf proto-oncogene serine/threonine kinase (BRAF) were higher in proximal tumors, and the mutation rates of adenomatous polyposis coli (APC), tumor protein 53 (TP53), and neuroblastoma RAS viral oncogene homolog (NRAS) were higher in distal tumors. The better RFS with the PI3K pathway mutation was significant only for proximal tumors, and the worse RFS with the RTK-RAS pathway mutation was significant only for distal tumors. CONCLUSIONS: A PI3K pathway mutation was associated with better RFS for CRC patients treated with adjuvant chemotherapy, and an RTK-RAS pathway mutation was associated with worse RFS. The significance of the prognostic impact differed according to the tumor location. Cancer 2017;123:3513-23. © 2017 American Cancer Society.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Regulação Neoplásica da Expressão Gênica , Mutação , Proto-Oncogenes/genética , Adulto , Idoso , Quimioterapia Adjuvante , Estudos de Coortes , Colectomia/métodos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Terapia Combinada , Intervalos de Confiança , Procedimentos Clínicos , Receptores ErbB/genética , Feminino , Genes ras , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Valor Preditivo dos Testes , Prognóstico , Proto-Oncogene Mas , República da Coreia , Estudos Retrospectivos , Análise de Sobrevida , Resultado do Tratamento
4.
Nucleic Acids Res ; 40(18): e140, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22705793

RESUMO

We developed a highly scalable 'shotgun' DNA synthesis technology by utilizing microchip oligonucleotides, shotgun assembly and next-generation sequencing technology. A pool of microchip oligonucleotides targeting a penicillin biosynthetic gene cluster were assembled into numerous random fragments, and tagged with 20 bp degenerate barcode primer pairs. An optimal set of error-free fragments were identified by high-throughput DNA sequencing, selectively amplified using the barcode sequences, and successfully assembled into the target gene cluster.


Assuntos
DNA/biossíntese , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência com Séries de Oligonucleotídeos , Penicilinas/biossíntese , Reação em Cadeia da Polimerase , Biologia Sintética
5.
Epigenomics ; 14(10): 615-628, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35473295

RESUMO

Aim: To construct a targeted bisulfite sequencing panel predicting origin of cancer of unknown primary. Methods: A bisulfite sequencing panel targeting 2793 tissue-specific markers was performed in 100 clinical samples. Results: The authors' prediction model showed 0.85 accuracy for the 'first-ranked' tissue type and 0.93 accuracy for the 'second-ranked' tissue type using 2793 tissue-specific markers and 0.84 accuracy for the 'first-ranked' tissue type and 0.92 accuracy for the 'second-ranked' tissue type when the number of tissue-specific markers was reduced to 514. Conclusion: Targeted bisulfite sequencing is a useful method for predicting the tissue of origin in patients with cancer of unknown primary.


When patients with cancer present with tumors that have migrated from elsewhere in the body, it is difficult for clinicians to identify where the cancer originated. DNA methylation profiling is a promising test to help identify where the cancer originated because it reflects cell of origin and is compatible with formalin-fixed, paraffin-embedded tissues. Because next-generation sequencing has already been implemented in clinical laboratories, the authors developed a targeted bisulfite sequencing panel that could predict the tissue of origin using genomic DNA extracted from formalin-fixed, paraffin-embedded tissues. The authors found that a hybrid capture-based targeted bisulfite sequencing panel is a useful method for predicting the tissue of origin in patients with cancer of unknown primary origin in clinical practice.


Assuntos
Neoplasias Primárias Desconhecidas , Metilação de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Primárias Desconhecidas/genética , Análise de Sequência de DNA/métodos , Sulfitos
6.
J Atheroscler Thromb ; 29(8): 1176-1187, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34456200

RESUMO

AIMS: Familial hypercholesterolemia (FH) is currently a worldwide health issue. Understanding the characteristics of patients is important for proper diagnosis and treatment. This study aimed to analyze the phenotypic and genetic features, including threshold cholesterol levels, of Korean patients with FH. METHODS: A total of 296 patients enrolled in the Korean FH registry were included, according to the following criteria: low-density lipoprotein-cholesterol (LDL-C) >190 mg/dL with tendon xanthoma or family history compatible with FH, or LDL-C >225 mg/dL. DNA sequences of three FH-associated genes were obtained using whole-exome or target exome sequencing. Threshold cholesterol levels for differentiating patients with FH/pathogenic variant (PV) carriers and predictors of PVs were identified. RESULTS: Of the 296 patients, 104 had PVs and showed more obvious clinical findings, including higher cholesterol levels. PV rates ranged from 30% to 64% when patients were categorized by possible or definite type according to the Simon Broome criteria. Frequent PV types included missense variants and copy number variations (CNVs), while the most frequent location of PVs was p.P685L in LDLR. The threshold LDL-C levels for patient differentiation and PV prediction were 177 and 225 mg/dL, respectively. Younger age, tendon xanthoma, and higher LDL-C levels were identified as independent predictors of PVs, while traditional cardiovascular risk factors were predictors of coronary artery disease. CONCLUSIONS: Korean patients with FH had variable PV rates depending on diagnostic criteria and distinctive PV locations. The reported threshold LDL-C levels pave the way for efficient patient care in this population.


Assuntos
Hiperlipoproteinemia Tipo II , Xantomatose , LDL-Colesterol/genética , Variações do Número de Cópias de DNA , Humanos , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/epidemiologia , Hiperlipoproteinemia Tipo II/genética , Mutação , Fenótipo , Receptores de LDL/genética , Sistema de Registros , República da Coreia/epidemiologia , Xantomatose/epidemiologia , Xantomatose/genética
7.
Anal Biochem ; 415(2): 218-20, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21536013

RESUMO

Here we present multiple target loci assembly sequencing (mTAS), a method for examining multiple genomic loci in a single DNA sequencing read. The key to the success of mTAS target sequencing is the uniform amplification of multiple target genomic loci into a single DNA fragment using polymerase cycling assembly (PCA). Using this strategy, we successfully collected multiloci sequence information from a single DNA sequencing run. We applied mTAS to examine 29 different sets of human genomic loci, each containing from 2 to 11 single-nucleotide polymorphisms (SNP) present at different exons. We believe mTAS can be used to reduce the cost of Sanger sequencing-based genetic analysis.


Assuntos
Loci Gênicos , Análise de Sequência de DNA/métodos , Éxons , Genoma Humano , Humanos , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único
8.
PLoS One ; 16(2): e0246356, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33524032

RESUMO

In the present study, we developed a computational method and panel markers to assess microsatellite instability (MSI) using a targeted next-generation sequencing (NGS) platform and compared the performance of our computational method, mSILICO, with that of mSINGS to detect MSI in CRCs. We evaluated 13 CRC cell lines, 84 fresh and 119 formalin-fixed CRC tissues (including 61 MSI-high CRCs and 155 microsatellite-stable CRCs) and tested the classification performance of the two methods on 23, 230, and 3,154 microsatellite markers. For the fresh tissue and cell line samples, mSILICO showed a sensitivity of 100% and a specificity of 100%, regardless of the number of panel markers, whereas for the formalin-fixed tissue samples, mSILICO exhibited a sensitivity of up to 100% and a specificity of up to 100% with three differently sized panels ranging from 23 to 3154. These results were similar to those of mSINGS. With the application of mSILICO, the small panel of 23 markers had a sensitivity of ≥95% and a specificity of 100% in cell lines/fresh tissues and formalin-fixed tissues of CRC. In conclusion, we developed a new computational method and microsatellite marker panels for the determination of MSI that does not require paired normal tissues. A small panel could be integrated into the targeted NGS panel for the concurrent analysis of single nucleotide variations and MSI.


Assuntos
Neoplasias Colorretais/genética , Instabilidade de Microssatélites , Linhagem Celular Tumoral , Predisposição Genética para Doença/genética , Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Repetições de Microssatélites/genética , Sensibilidade e Especificidade
9.
Sci Rep ; 11(1): 16333, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381078

RESUMO

Circulating tumor DNA (ctDNA) may reveal dynamic tumor status during therapy. We conducted serial ctDNA analysis to investigate potential association with clinical outcome in metastatic colorectal cancer (mCRC) patients receiving chemotherapy. Tissue KRAS/NRAS wild-type mCRC patients were enrolled and treated with first-line cetuximab-containing chemotherapy. ctDNA isolated from plasma were analyzed by next generation sequencing (NGS) with 16 targeted gene panel. Among 93 patients, 84 (90.3%) had at least 1 somatic mutation in baseline ctDNA samples (average 2.74). Five patients with KRAS or NRAS hotspot mutation in the ctDNA showed significantly worse progression-free survival (PFS) (p = 0.029). Changes in average variant allele frequency (VAF) in ctDNA showed significant correlation with tumor size change at the time of first response evaluation (p = 0.020) and progressive disease (PD) (p = 0.042). Patients whose average VAF decreased below cutoff (< 1%) at the first evaluation showed significantly better PFS (p < 0.001), and the average VAF change further discriminated the PFS in the patients in partial response (p = 0.018). At the time of PD, 54 new mutations including KRAS and MAP2K1 emerged in ctDNA. ctDNA sequencing can provide mutation profile that could better reflect tumor mutation status and predict treatment outcome.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , DNA Tumoral Circulante/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Imunológicos/uso terapêutico , Cetuximab/uso terapêutico , DNA Tumoral Circulante/sangue , Neoplasias Colorretais/sangue , Receptores ErbB/metabolismo , Feminino , Frequência do Gene/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/efeitos dos fármacos , Intervalo Livre de Progressão , Proteínas ras/genética
10.
Chembiochem ; 11(17): 2448-52, 2010 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-20981747

RESUMO

The fundamental problem for low-cost gene synthesis is errors that occur during the synthetic process. To address this problem, we developed a practical method that exploits the fact that the predominant errors are deletions. In this method, a simple fluorescence-based readout was used to distinguish error-free synthetic DNA molecules. To do this, we constructed vectors that contained multiple cloning sites and GFP. In the vectors, the GFP gene is designed to be out-of-frame, but insertion of an in-framed synthetic DNA construct into the appropriate cloning site will lead to fluorescent cell colonies. We successfully used this method to synthesize five genes and improved the bp per error from 629 to 6552 by selecting green fluorescent colonies.


Assuntos
Fluorescência , Genes Sintéticos/genética , Proteínas de Fluorescência Verde/genética , Sequência de Bases , DNA/genética , DNA Polimerase Dirigida por DNA/genética , Técnicas Genéticas , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Reprodutibilidade dos Testes
11.
Clin Cancer Res ; 25(20): 6141-6147, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31285374

RESUMO

PURPOSE: Recent sequencing studies revealed that a subset of colorectal cancer harbors a significantly higher number of somatic mutations. These hypermutated tumors show distinct clinicopathologic features. However, the prognostic impact of the hypermutated tumors is not clearly established. EXPERIMENTAL DESIGN: We analyzed tumor mutation burden (TMB) from targeted next-generation sequencing data of 40 major genes in 516 patients with colorectal cancer. TMB was defined as total number of nonsynonymous mutations per tumor. Cutoff value for TMB-high was chosen by which best discriminated relapse-free survival (RFS) using the Contal and O'Quigley method. RESULTS: In the TCGA data, mutation count of the selected 40 genes reflected the whole exome mutation burden (Pearson correlation = 0.873, P < 0.001). In our patient cohort, 8 or more mutations in the 40 genes was defined as TMB-high, which best discriminated RFS. A total of 55 patients (10.7%) had TMB-high. TMB-high tumors were more frequently found in a proximal location (63.6%) and had a higher proportion of N0 disease (30.9%) and MSI-H (49.1%) compared with TMB-low. Most importantly, TMB-high was associated with better 5-year RFS compared with TMB-low (96.3% vs. 79.8%, P = 0.005). Although there was significant overlap between TMB-high and MSI-H, MSI-H status was not significantly associated with RFS. Multivariate analysis revealed TMB-high as an independent positive prognostic factor for RFS [adjusted HR, 0.16 (95% confidence interval, 0.04-0.66), P = 0.011]. CONCLUSIONS: TMB-high is associated with better prognosis in patients with colorectal cancer treated with curative surgery followed by adjuvant fluoropyrimidine and oxaliplatin chemotherapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Capecitabina/uso terapêutico , Neoplasias Colorretais/genética , Recidiva Local de Neoplasia/epidemiologia , Oxaloacetatos/uso terapêutico , Idoso , Quimioterapia Adjuvante/métodos , Colo/patologia , Colo/cirurgia , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Análise Mutacional de DNA , Intervalo Livre de Doença , Feminino , Fluoruracila/uso terapêutico , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucovorina/uso terapêutico , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Mutação , Recidiva Local de Neoplasia/genética , Estadiamento de Neoplasias , Compostos Organoplatínicos/uso terapêutico , Prognóstico , Reto/patologia , Reto/cirurgia , Estudos Retrospectivos
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