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随着处理基因表达等应用的新技术和方法的出现,很难确定哪种方法更好,一种方法是否可以简单地代替另一种方法,或者搭配使用能否提供更完美的结果?在此,我们尝试阐明如何在实时荧光定量 PCR (qPCR) 和下一代测序 (NGS) 之间进行选择,或者您是否真的需要择其一。
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全转录组 NGS 有两种主要方法:RNA-Seq 和靶向转录组。当以下一个或多个项目对于您的研究工作至关重要时,建议使用 RNA-Seq:
靶向转录组方法有相似之处;尽管输出将限于较常见的 mRNA 转录本,但在涵盖大多数已知转录组的20,000个靶标范围内,通常可能有新发现。
实时荧光定量 PCR 在哪些方面适合以下工作流程?数据完整性是基因表达实验的关键,因此普遍做法是在 NGS 的上游和下游均使用实时荧光定量 PCR。这意味着,我们并非必须在实时荧光定量 PCR 与 NGS 中择其一;恰恰相反,这两种技术是互补的,共同使用可以生成值得信赖的结果。在 NGS 的上游,通常使用 TaqMan 实时荧光定量 PCR 来检查 NGS 前 cDNA 的完整性。
在 NGS 的下游,实时荧光定量 PCR 仍然是结果验证的必备方法。若不需要进行验证,务必注意实时荧光定量 PCR 是开展涉及在 NGS 筛选过程中发现的转录本靶向组合的后续研究的金标准技术。
下列出版物重点介绍了 Ion AmpliSeq NGS 与 TaqMan 实时荧光定量 PCR 在 cDNA 完整性检查或结果验证方面的共同应用。
阅读我们表明 TaqMan 基因表达测定、Clariom D 测定和 Ion AmpliSeq 转录组试剂盒之间高度一致的技术资料。
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Circulating tumor cell plasticity determines breast cancer therapy resistance via neuregulin 1-HER3 signaling. Nature cancer | 2025 Jan 03 | PubMed ID: 39753722 | Read Article "...t recurrent mutations51. Library preparation and sequencing were performed using multiplex PCR-based Ion Torrent AmpliSeq (Thermo Fisher Scientific) and Ion S5XL technology, as previously described52...""..ntin (Agilent Dako, clone M7020, 1:1,000). For HER3 expression, we incubated samples with anti-HER3/ ERBB3 (D22C5, XP rabbit monoclonal 12708, 1:50) at 4 °C overnight and used heat-induced antigen unmasking with damp heat at 90 °C with EDTA unmasking solution (pH 9 (1:10); 14747 Signal Stain) for 30 min. Sections were scanned using a Zeiss AxioScan, and representative images are shown.
Targeted next-generation sequencing of somatic mutations Genomic DNA was extracted from CDOs and CDXs. DNA concentration was assessed by fluorimetric measurement using a QuBit 3.0, and the amount of amplifiable DNA (sequencing-grade quality) was determined using a quantitative assay (TaqMan RNaseP detection assay) on a StepOnePlus instrument (both Thermo Fisher Scientific). Samples were amplified using a custom-designed gene panel for BC50, covering the most recurrent mutations51. Library preparation and sequencing were performed using multiplex PCR-based Ion Torrent AmpliSeq (Thermo Fisher Scientific) and Ion S5XL technology, as previously described52..." More... | |
Anti-IL-4Rα aptamer reduces IL-4 signaling and formation of nasal polyps ERJ Open Research | 2024 Nov 14 | Read Article "...lyp tissues using RNeasy Kits (Qiagen). We
used 1 ng of RNA for targeted whole RNA-seq with the AmpliSeq whole transcriptome on the S5 system (Thermo Fisher Scientific). Barcoded cDNA libraries were p...""..orgDownloaded from
was used for evaluating changes in gene expression and protein levels. The gene expression level was measured for IL-4R. Flow cytometry was used to measure the expression levels of cell surface IL-4R and intracellular IL-4 cytokine. The protein levels
of IL-4R, STAT6, p-STAT6, and GATA3 were measured by western blot assays.
RNA Extraction from nasal polyp tissue Total RNA was extracted from the nasal polyp tissues using RNeasy Kits (Qiagen). We
used 1 ng of RNA for targeted whole RNA-seq with the AmpliSeq whole transcriptome on the S5 system (Thermo Fisher Scientific). Barcoded cDNA libraries were prepared using
the SuperScript VILO cDNA synthesis kit (Invitrogen) and amplified with the Ion AmpliSeq Transcriptome Human Gene Expression kit (Thermo Fisher Scientific) to create
the RNA-seq library. The quality of the cDNA libraries was assessed using the TaqMan library quantitation kit (Applied Biosystems). After diluting the libraries to 100 pM, we
pooled them together and performed emulsion PCR amplification on Ion O.." More... | |
Characterizing the Mutational Landscape of Diffuse Large B-Cell Lymphoma in a Prospective Cohort of Mexican Patients International Journal of Molecular Sciences | 2024 Aug 28 | Read Article "...sion, diagnosis, and treatment response of lymphomas according to the literature, using the Ion AmpliSeq Chef library kit. Pools of eight samples were loaded onto Chip Ion 550 (Thermo Fisher Scientifi...""..A glycosylase (Thermo Fisher Scientific) plus 20 µL of water and incubated (37 °C × 2 min and 50 °C × 10 min); 10 ng of each sample was used for the libraries. For analyses, a comprehensive literature review was undertaken to identify publications featuring genomic analyses that categorized DLBCL patients into distinct clusters [ 9 , 10 , 11 ]. After the literature review, a custom panel was meticulously designed, comprising genes. This was consistently and repeatedly reported by these authors. This custom panel (6026 oligos) was used for the sequencing of coding regions and splicing sites of 79 genes ( Supplementary Table S1 ) associated with the progression, diagnosis, and treatment response of lymphomas according to the literature, using the Ion AmpliSeq Chef library kit. Pools of eight samples were loaded onto Chip Ion 550 (Thermo Fisher Scientific). Then, the libraries were quantified by qPCR with the TaqMan RNase P detection kit (Thermo Fisher Scientific) and sequenced using the Ion GeneStudio S5 Prime System platform v. 5.18. .." More... | |
Changes in mutations of cell-free DNA and liver tumor tissue in patients with advanced hepatocellular carcinoma before and after introduction of lenvatinib. Oncology | 2024 Jul 24 | PubMed ID: 39047713 | Read Article "...) for analysis of the CTNNB1 and TP53 mutations. Specifically, according to the protocol of the AmpliSeq library, PCR was performed to amplify the required region first, the ligation next, and purific...""..d ABI QuantStudio 3D (Thermo Fisher Scientific, Massachusetts, USA) to run the digital PCR [25]. We ordered a dedicated master mix and assay in advance (TaqMan Liquid Biopsy dPCR Assay), then performed digital PCR on - 124C>T and -146C>T mutations, which are the most common and well-known ones [26, 16, 27].
Analysis of CTNNB1 and TP53 with next-generation sequencing
We chose Ion Torrent Next-Generation Sequencing Technology (Thermo Fisher Scientific) for analysis of the CTNNB1 and TP53 mutations. Specifically, according to the protocol of the AmpliSeq library, PCR was performed to amplify the required region first, the ligation next, and purification was performed using Agencourt AMPure XP Reagent. After that, we adjusted templates using Ion One Touch 2 system and Ion One Touch ES. Finally, we ran NGS on the Ion Personal Genome Machine (Ion PGM) system. Supplementary table 1 shows information on amplicon regions amplified by NGS. For confirmation, we used The Integrative Genomics Viewer (IGV) 2.12.3 to visualize the genomic data [28]..." More... | |
Successive next-generation sequencing strategy for optimal fusion gene detection in non-small-cell lung cancer in clinical practice. Pathology | 2024 Jul 05 | PubMed ID: 38834439 | Read Article "...d in parallel using targeted DNA and RNA NGS panels. DNA sequencing was performed using the Ion AmpliSeq Colon-Lung Cancer Research PanelV2 (ThermoFisher Scientific), covering >500 hotspot mutations i...""..Tumour mutation testing followed a two-step algorithm. Samples were analysed using TaqMan probes (ThermoFisher Scientific) for rapid identification of EGFR and KRAS frequent mutations.27 Mutated samples underwent targeted DNA NGS panel exclusively while EGFR and KRAS wildtype samples were assessed in parallel using targeted DNA and RNA NGS panels. DNA sequencing was performed using the Ion AmpliSeq Colon-Lung Cancer Research PanelV2 (ThermoFisher Scientific), covering >500 hotspot mutations in KRAS, EGFR, BRAF, PIK3CA, AKT1, ERBB2, PTEN, NRAS, STK11, MAP2K1, ALK, DDR2, CTNNB1, MET, TP53, SMAD4, FBXW7, NOTCH1, ERBB4, FGFR1, FGFR2, FGFR3. Multiplex PCR libraries were prepared using 30 ng of DNA whenever possible and 3 mL of DNA for samples with DNA concentration <10 ng/mL by AmpliSeq technology (Ion AmpliSeq library kit V2) following the manufacturer’s protocol. Variant call files from the variant caller were loaded on a galaxy platform19 and annotated using the Safir2report tool.27,28
RNA analysis: gene fusion detection
Gene fusion det.." More... | |
LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue Heliyon | 2024 Jun 12 | PubMed ID: 38988576 | Read Article "...Whole transcriptome library was prepared from both unamplified and amplified RNA using the Ion AmpliSeq human gene expression on S5 system (Thermo Fisher Scientific, USA) as previously described [ 51 ...""Whole transcriptome library was prepared from both unamplified and amplified RNA using the Ion AmpliSeq human gene expression on S5 system (Thermo Fisher Scientific, USA) as previously described [ 51 ]. For each FFPE sample, ∼10 ng of gDNA-free RNA was used to prepare barcoded libraries using Ion AmpliSeq transcriptome human gene expression kit (Thermo Fisher Scientific, USA). Purified barcoded libraries were quantified using TaqMan library quantitation kit (Applied Biosystems). The libraries were diluted to 100 pM, pooled together, amplified using emulsion PCR on the Ion One Touch 2 (OT2) instrument, and enriched using the Ion One Touch ES as per manufacturer's instructions. RNA-sequencing of the libraries was performed using Ion S5 XL Semiconductor sequencer on Ion 540 Chip (Thermo Fisher Scientific, USA) as previously described [ 51 ]. " More... | |
Modelling atopic dermatitis in healthy human skin for the characterization of topical compounds. Experimental dermatology | 2024 May 25 | PubMed ID: 38794814 | Read Article "... instruction. The internal controls were B2M and RPLP0 (Life technologies).
Whole transcriptome AmpliSeq analysis was performed by reverse transcription of 10 ng total RNA in 5 μL, followed by an ampl...""..NA was prepared using RNeasy Mini kit (Qiagen) as recommended by the manufacturer and was collected in 30 μL of RNase- free water. The concentration of RNA was measured with a NanoDrop2000 instrument, and the high quality was confirmed by Agilent 2100 Bioanalyzer. Samples were stored at −80°C until use. Quantitative PCR was performed using the TaqMan RNA- to- CT 1- Step kit
(Applied Biosystems) according to the supplier's instruction. The internal controls were B2M and RPLP0 (Life technologies).
Whole transcriptome AmpliSeq analysis was performed by reverse transcription of 10 ng total RNA in 5 μL, followed by an amplification of 23 930 RefSeq gene transcript cDNAs by ultra- high multiplexed PCR in 20 μL using the Ion AmpliSeq Transcriptome Gene Expression Kit (ThermoFisher) and IonCode barcodes. After construction of sequencing libraries, the unamplified libraries were quantified by qPCR, and equal molar amounts were combined for sequencing. Templating of Ion Sphere Particles and loading onto Ion 550 chips was automatically conducted.." More... | |
Quality-Assured Analysis of PIK3CA Mutations in Hormone Receptor-Positive/Human Epidermal Growth Factor Receptor 2-Negative Breast Cancer Tissue: A Story About the Need for Proficiency Testing for High-Quality Molecular Biomarker Reporting in Precision Medicine. The Journal of molecular diagnostics : JMD | 2024 Apr 30 | PubMed ID: 38697471 | Read Article "...ck Biotech OncoScreen ParpMatch for Tissue Kit, HS
1 1 (100)
OncoScreen Plus 1 1 (100) Illumina AmpliSeq Cancer HotSpot
Panel version 2 for Illumina
1 0 (0)
Qiagen GeneRead QIAact Actionable Insights ...""..on Test
3 3 (100)
Thermo Fisher Scientific Commercial and custommade TaqMan assay: c.1633G>A, c.1258T>C, c.1635G>T, c.3140A>T, and c.1624G>A
1 0 (0)
Next-generation sequencingQ33
e In-house primer 1 1 (100) e Custom-designed probes
that cover approximately 1.1 Mb of genomic sequences for 1021 cancer-related genes
1 1 (100)
e BGI Lung Cancer Genetic Test
1 1 (100)
AmoyDx AmoyDx HANDLE Classic NGS Panel
1 1 (100)
Burning Rock Biotech OncoScreen ParpMatch for Tissue Kit, HS
1 1 (100)
OncoScreen Plus 1 1 (100) Illumina AmpliSeq Cancer HotSpot
Panel version 2 for Illumina
1 0 (0)
Qiagen GeneRead QIAact Actionable Insights Tumor Panel
1 1 (100)
QIAseq Targeted DNA Human Actionable Solid Tumor Panel
1 0 (0)
Thermo Fisher Scientific Oncomine Focus Assay 3 3 (100) Ion AmpliSeq Library Kit Plus; in-house primer 1 1 (100)
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Multi-UniFocality (MUF), in contrast to multifocality, in thyroid lesions: Relation to lymphocytic thyroiditis. Pathology international | 2024 Apr 01 | PubMed ID: 38558427 | Read Article "...RNA isolation system.17
Molecular diagnostics using NGS for somatic gene variant (with a custom AmpliSeq™ Cancer Hotspot Panel; Thermo Fisher Scientific) and/or gene fusion analysis (with Archer® Fusi...""..ysis, material from histological slides or occasionally cytology was morphologically selected by an experienced pathologist subspecialized in thyroid pathology.16 Molecular analyses were performed as part of the routine diagnostic workup in the Molecular Diagnostics Unit of the Pathology department (ISO15189 accredited) at the Leiden University Medical Center (LUMC). Nucleic acid was purified using a fully automated DNA/ RNA isolation system.17
Molecular diagnostics using NGS for somatic gene variant (with a custom AmpliSeq™ Cancer Hotspot Panel; Thermo Fisher Scientific) and/or gene fusion analysis (with Archer® FusionPlex® CTL panel; ArcherDX Inc.) were described previously.18–20 From 2013 through 2015/2016, a hotspot mutation analysis using Taqman hydrolysis assay was used.21 From 2015, a custom AmpliSeq Cancer Hotspot Panel (CHSP) was used with frequent updates. The CHSPv2/v3/v4/v6 targets 50/60/ 74/85 genes, respectively. The Archer FusionPlex CTL panel targeted 36 genes and was used from 2016 on. Molecular diagnostic results wer.." More... | |
Characterization and Distribution of SARS-CoV-2 Omicron Variant and its Sub-lineages in Uttarakhand using Next Generation Sequencing: A Retrospective Study Journal of Pure and Applied Microbiology | 2024 Mar 01 | Read Article "...Ion AmpliSeqTM Library kit Plus (Thermo Fisher Scientific, Waltham, Massachusetts, United States) were used for library preparation according to manufacturer’s instruction. First the samples were isol...""..Ion AmpliSeqTM Library kit Plus (Thermo Fisher Scientific, Waltham, Massachusetts, United States) were used for library preparation according to manufacturer’s instruction. First the samples were isolated and quantification of viral RNA was done. For Ion AmpliSeqTM SARS-CoV-2 Research panel library (Thermo Fisher Scientific, Waltham, Massachusetts, United States), sample containing as little as 20 copies of viral RNA (10 copies per target amplification reaction) was used. This research panel consists of two 5X primer pair pools that targets 23 amplicon which are specific to the SARS-CoV-2 coronavirus, and 5 human expression controls. The panel with amplicon length of range 125-25 bp provides almost 99% coverage of SARS-CoV-2 genome and covers all potential serotypes. MagMAXTM Viral/Patogen Nucleic Acid Isolation Kit (Thermo Fisher Scientific, Waltham, Massachusetts, United States) was used for RNA isolation. TaqManTM 2019-nCoV Assay Kit v1, TaqManTM 2019-nCoV Control Kit v1, and TaqPathTM 1-step RT-qPCR master Mix (Thermo Fisher Scien.." More... | |
Genomic surveillance of malaria parasites in an indigenous community in the Peruvian Amazon ppr0812746 | 2024 Feb 29 | Read Article "...ected by each study site. These procedures were done up to one week before the sequencing runs. AmpliSeq Assays Pv and Pf AmpliSeq Peru library preparation was performed as previously described11,13,2...""..ugemont assay were used in a SYBR Green-based qPCR. After that, the TaqMan assay was used only with positive infections in the rst reaction. Pv and Pf positive samples selected from the studies described above were processed again to ensure DNA quality for the sequencing. DNA was re-extracted using the EZNA® Blood DNA Mini kit, and the Mangold protocol23 was performed. Samples with parasitemia > 5 par/µl were randomly selected by each study site. These procedures were done up to one week before the sequencing runs. AmpliSeq Assays Pv and Pf AmpliSeq Peru library preparation was performed as previously described11,13,26 using the AmpliSeq Library PLUS kit (Illumina). Brie y, each sample (7.5 µl of DNA) was ampli ed by PCR using two different sets of primer panels. Then, the PCR products were mixed and partially digested with the FuPa reagent, and indexes were ligated. Next, a washing step was performed with Agencourt AMPure XP beads (Beckman Coulter). Once ready, the library was ampli ed by PCR and washed again to remove genomic DNA an.." More... | |
Quantitation of human mitochondrial DNA and whole mtGenomes sequencing of fingernail/hair shaft samples Forensic Sciences Research | 2024 Feb 22 | Read Article "...amplicons in two multiplex pools with an average targeted fragment size of 163 bp using the Ion AmpliSeq technology (Thermo Fisher Scientific). The purpose of this study was to establish this reliable...""..hole mtGenome sequencing. For mtDNA quantification, we designed the primers and TaqMan probe to construct mtDNA-specific standard curves by using real-time PCR, because mtDNA commercial standard substance was rare (NIST has recently made a Human DNA Quantitation Standard with mtDNA quantity as a non-certified value, presented as a ratio of mtDNA to nuclear DNA quantity [22, 23]). For whole mtGenome sequencing, MPS was conducted on the Ion Torrent Personal Genome Machine system (PGM; Thermo Fisher Scientific, Foster City, CA, USA). Considering the low concentration and short fragments of fingernail/hair shaft samples, the Precision ID mtDNA panel (Thermo Fisher Scientific) was selected for this work. The multiplex PCR reaction comprised 162 amplicons in two multiplex pools with an average targeted fragment size of 163 bp using the Ion AmpliSeq technology (Thermo Fisher Scientific). The purpose of this study was to establish this reliable mtDNA profile method for fingernail/hair shaft samples and evaluate its forensic application value..." More... | |
Haplotype phasing of CYP2D6 : an allelic ratio method using Agena MassARRAY data Translational Psychiatry | 2024 Feb 12 | PubMed ID: 38346976 | Read Article "...ays for CYP2D6 , the components of the previously available Luminex xTAG CYP2D6 v3 kit, the Ion Ampliseq Pharmacogenomics Panel, PharmacoScan Solution, Agena Bioscience assays such as the Veridose Cor...""..ical Pharmacogenetics Implementation Consortium (CPIC), the Dutch Pharmacogenetics Working Group (DPWG), the Canadian Pharmacogenomics Network for Drug Safety (CPNDS) and the French National Network (Réseau) of Pharmacogenetics (RNPGx) [ 29 – 31 ]. There are a variety of genetic technologies that aim to identify CYP2D6 haplotypes. These include TaqMan Single Nucleotide Polymorphism (SNP) and Copy Number Variant (CNV) assays for CYP2D6 , the components of the previously available Luminex xTAG CYP2D6 v3 kit, the Ion Ampliseq Pharmacogenomics Panel, PharmacoScan Solution, Agena Bioscience assays such as the Veridose Core Panel, and digital PCR [ 32 – 38 ]. However, none of these claimed to be able to conduct haplotype phasing for CYP2D6 . There is one assay that was previously available and able to conduct haplotype phasing for a number of CYP2D6XNs (duplications/multiplications, specifically *1 , *2 , *4 , *10 , *17 , *35 , and *41 ), the AmpliChip CYP450 Test [ 32 ]. Although this assay had this capability, it ceased to be supported a.." More... | |
Comparison of analytical performance and economic value of two biosurveillance methods for tracking SARS-COV-2 variants of concern. Microbiology spectrum | 2024 Feb 08 | PubMed ID: 38206048 | Read Article "... RT-PCR. Ion Torrent WGS consumables and reagents included: SuperScript IV VILO Master Mix, Ion Ampliseq Library Kit Plus and SARS-CoV-2 Insight Research Assay, Ion 540 Kit-Chef, and Qubit 4 Fluoromet...""..al cost of instrumen tation, (2) cost of consumables and reagents, and (3) cost of staffing rates multiplied by the hours of required hands-on-time. Ion Torrent WGS instrumentation included the following: QuantStudio 6 Pro RT-PCR, Ion Chef chip templating system, Ion Torrent GeneStudio S5 Plus sequencer, and Ion PGM Torrent Server. TaqMan Mutation Panel/ genotyping instrumentation included the following: QuantStudio 6 Pro RT-PCR. Ion Torrent WGS consumables and reagents included: SuperScript IV VILO Master Mix, Ion Ampliseq Library Kit Plus and SARS-CoV-2 Insight Research Assay, Ion 540 Kit-Chef, and Qubit 4 Fluorometer and Qubit dsDNA HS Assay Kit. TaqMan Mutation Panel/genotyping consumables and reagents included the following: TaqPath 1-Step RT-qPCR Master Mix, CG, and the eight or twelve TaqMan SARS-CoV-2 Mutation Panel assays used in the 8-assay and 12-assay mutation panel layouts. Rates for staffing and hands-on-time were calculated based on the average hourly rate of LSTC clinical full-time employees (FTE), with Ion Torrent WGS.." More... | |
Detection of SARS-CoV-2 B.1.1.529 (Omicron) variant by SYBR Green-based RT-qPCR Biology Methods & Protocols | 2024 Jan 01 | Read Article "...kit is 800 copies/mL when Ct value of ORF1ab is 34 and is 1560 copies/ml when its Ct is 33 (Ion AmpliSeq SARS CoV 2 Research Panel SARS-CoV-2). So, the LOD of our protocol is between 800 and 1560 copi...""..using the serial dilution results. Linear regression performed for these primer sets demonstrated strong correlation ( Fig. 2 ). A limit of detection (LOD) was determined for each primer set and identified as 0.0005 ng/µL for the two sets ( Table 2 , Fig. 2 ), or with maximal Ct of 34.05 or 35.5 obtained by TaqMan when targeting ORF1ab and SYBR Green RT-PCR, respectively. According to the manufacturer, the LOD of TaqPath kit is 800 copies/mL when Ct value of ORF1ab is 34 and is 1560 copies/ml when its Ct is 33 (Ion AmpliSeq SARS CoV 2 Research Panel SARS-CoV-2). So, the LOD of our protocol is between 800 and 1560 copies/ml. Moreover, the specificity of our primers was investigated by four different ways: (i) using 15 clinical positive samples for SARS-CoV-2 confirmed Delta variant; (ii) using 100 clinical negative samples for SARS-CoV-2 by TaqPath kit; they did not manifest a signal when tested with such negative controls; (iii) using in silico prediction analyses as described in the Material and Methods section; and (iv) by WGS of 1.." More... | |
Pharmacogenetic and clinical predictors of voriconazole concentration in hematopoietic stem cell transplant recipients receiving CYP2C19-guided dosing. The pharmacogenomics journal | 2023 Nov 22 | PubMed ID: 37925536 | Read Article "...s2860840C>T (Assay ID C__11201742_10) and rs11188059G>A (Assay ID C__31983321_10). A custom Ion AmpliSeq Pharmacogenetics Panel (Thermo Fisher Scientific, Waltham, MA) was used to batch genotype for S..."".. results were mapped to star allele nomenclature using TaqMan AlleleTyper Software together with translation tables developed from established guidelines as set forth by the CPIC. The turnaround time for genotyping was 48–72 h and results were available prior to the first dose of voriconazole. CYP2C genotyping was performed as described for CYP2C19 with the exception that TaqMan DME Genotyping Assays were used to detect rs2860840C>T (Assay ID C__11201742_10) and rs11188059G>A (Assay ID C__31983321_10). A custom Ion AmpliSeq Pharmacogenetics Panel (Thermo Fisher Scientific, Waltham, MA) was used to batch genotype for SNPs in ABCB1 (1236G > A, 2677C > T/A, 3435G > A), ABCG2 (421G > T), CYP2C9 (*2, *3, *4, *5, *6, *8, *9, *10, *11, *13, *15), CYP3A4 (*1B, *22), and CYP3A5 (*3, *6, *7). Sequencing libraries were prepared using an Ion Ampliseq Library Kit (Thermo Fisher Scientific) per manufacturer’s instructions. Briefly, amplicons are ligated to ion-compatible adapters, followed by nick repair to complete the linkage between adapters and.." More... | |
Resistance to mesenchymal reprogramming sustains clonal propagation in metastatic breast cancer. Cell reports | 2023 Oct 04 | PubMed ID: 37257449 | Read Article "...ancercovering themost recurrentmutations.Librarypreparationandsequencingwereperformedusing the multiplex PCR-based Ion Torrent AmpliSeqTM technology (Thermo Fisher Scientific) and Ion S5XL technology...""..cturer’s protocol), and anti-KRT5/6 (DAKO, D5/16 B4 clone, manufacturer’s protocol), anti-CD10 (Leica Biosystems, diluted 1:10), anti-human ZEB1 (Sigma Aldrich, rabbit polyclonal #HPA027524, diluted 1:100) anti-GATA3 (Becton Dickinson, clone L50-823, concentration 0.2 mg/L).
Targeted sequencing of somatic mutations GenomicDNAwasextracted fromFACS-purifiedcancer cells andmatchedgermlinecontrols (CD45+white bloodcells purifiedbyFACS sorting from the samemetastatic effusion). The concentration of nucleic acidswas assessedbyfluorimetricmeasurement throughQuBit 3.0 and the amount of amplifiable DNA (sequencing-grade quality) was determined using a quantitative assay (TaqMan RNAseP detection assay, Thermo Fisher Scientific) on a StepOnePlus device (Thermo Fisher Scientific). DNA samples were amplified using a customdesignedgenepanel for breastcancercovering themost recurrentmutations.Librarypreparationandsequencingwereperformedusing the multiplex PCR-based Ion Torrent AmpliSeqTM technology (Thermo Fisher Scientific) and Ion S5XL technology..." More... | |
Evaluation of loci to predict ear morphology using two SNaPshot assays. Forensic science, medicine, and pathology | 2023 Sep 26 | PubMed ID: 36401782 | Read Article "...o obtain genotypic data including TaqMan assays [18, 19], next-generation sequencing (NGS), Ion Ampliseq technology [20] and whole-genome sequencing (WGS) [21]. However, whole-genome sequencing is an ...""..ps (Europeans, Americans cohorts) identified 49 significant loci associations [4]. The genetic variations like SNPs insertion-deletion variants, block substitution and inversion variants may cause amino acid substitutions which alter the functional property of the protein [3]. This results in morphological changes and distinct phenotypes [17].
Previously developed phenotyping assays used a variety of reported techniques to obtain genotypic data including TaqMan assays [18, 19], next-generation sequencing (NGS), Ion Ampliseq technology [20] and whole-genome sequencing (WGS) [21]. However, whole-genome sequencing is an expensive technique and not suitable for the specific traits of interest involving limited genes. Multiplex analyses coupled with the mini sequencing technique offer a targeted approach for retrieval of specific phenotypes of interest [22–25]. The phenotypic variation in population caused by genetic variation must be added to modelling parameters [17]. Regression analyses are performed to model the structure (identify the.." More... | |
Gene fusions and gene variants associated with cancer us11746379 | 2023 Sep 05 | Read Article "...ing reaction, such as a next generation sequencing reaction. In these embodiments, for example, AmpliSEQ™ (Life Technologies/Ion Torrent, Carlsbad, Calif.) or TruSEQ™ (Illumina, San Diego, Calif.) tec..."".. aforementioned genes. The set of amplicons are detected by a set of matched probes. In an exemplary embodiment, the invention is a set of TaqMan™ (Roche Molecular Systems, Pleasanton, Calif.) assays that are used to detect a set of target genetic variations used in the methods of the invention.
In one embodiment, the set of probes are a set of primers used to generate amplicons that are detected by a nucleic acid sequencing reaction, such as a next generation sequencing reaction. In these embodiments, for example, AmpliSEQ™ (Life Technologies/Ion Torrent, Carlsbad, Calif.) or TruSEQ™ (Illumina, San Diego, Calif.) technology can be employed. In other embodiments, the two or more probes are primer pairs.
A modified ribonucleotide or deoxyribonucleotide refers to a molecule that can be used in place of naturally occurring bases in nucleic acid and includes, but is not limited to, modified purines and pyrimidines, minor bases, convertible nucleosides, structural analogs of purines and pyrimidines, labeled, derivatized and modified nucleo.." More... | |
Dynamics of SARS-CoV-2-Specific B Cell Memory Responses in Infected and Vaccinated Individuals. Viral immunology | 2023 Jun 19 | PubMed ID: 37140898 | Read Article "... some of SARS-CoV-2positive patients. The sequencing analysis of SARS-CoV-2 was performed using AmpliSeq SARS-CoV-2 panel (Thermo Fisher Scientific). The reads from the library were aligned with the W...""..n-A.
Afterward, double staining with two spike-tetramer solutions was used. Whenever further characterization of B cells was required, staining of cells using 10, flow cytometric data were analyzed with the MACSQuantify Software. Furthermore, we genotyped SARS-CoV-2 mutations using TaqMan Universal PCR Master Mix and specific custom TaqMan probe (Fabiani et al., 2022) and sequenced using nextgeneration sequencing (NGS) in some of SARS-CoV-2positive patients. The sequencing analysis of SARS-CoV-2 was performed using AmpliSeq SARS-CoV-2 panel (Thermo Fisher Scientific). The reads from the library were aligned with the Wuhan-Hu-1 NCBI Reference Genome (Accession Number: MN908947.3) in Torrent Suite v. 5.10.1. A multivariate linear regression model using the enter approach was
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applied for simultaneously assessing the effects of independent variables on a quantitative dependent variable; p < 0.05 was considered statisti.." More... | |
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Illumina 靶向扩增子 RNA-Seq 是一种 NGS 方法,可检测感兴趣的 12-1,200 个转录本。此靶标范围与使用实时荧光定量 PCR 可能得到的靶标范围重叠(图1)。在实时荧光定量 PCR 和 Illumina 靶向扩增子 RNA-Seq 之间选择时,应考虑以下因素:
可以,TaqMan 基因表达测定可用于我们预设计的测定系列中的所有基因和种属的大部分外显子-外显子连接。 要选择变体专用的测定,请先确定 NCBI RefSeq 转录本登录号,然后在 TaqMan 测定搜索工具中使用该登录号进行搜索。 在随后出现的测定列表中,选择仅检测感兴趣的那个变体的测定。 如果所有可用的测定均检测超过一个 RefSeq 转录本,则使用我们的定制测定设计工具尝试设计变体专用的测定。
有,TaqMan 阵列板是含有 8-384 种测定试剂(预点样并干燥,您只需添加 cDNA 和预混液)的96孔和384孔板。 根据所需的样本和靶标数量,订购适量的板来实现技术复制和生物学复制。 TaqMan 阵列卡是含有 12-384 种测定试剂的384孔微流控卡,其具有预混液用量比传统板少的附加优势,还具有超简单的台式工作流程:在2分钟内对384个孔进行移液。 TaqMan OpenArray 板是适用于 qPCR 的较高通量选项,每个数据点的价格较低。
事实上这得看情况。 一次 Illumina 靶向扩增子 RNA-Seq 运行可分析的样本和靶标数量取决于 Illumina 平台和化学试剂。 假定所需数量的样本和靶标适合一次 Illumina 靶向扩增子 RNA-Seq 运行,则从样本制备到数据分析的时间为两天。 如果您必须将 Illumina NGS 实验外包给核心机构或服务提供商,可能需要数天或数周才能获得结果。 与之相比,以下实时荧光定量 PCR 实验在 1-3 天内就可完成:
仅供科研使用,不可用于诊断目的。