The advantages and disadvantages of real-time PCR and NGS
As new technologies and methodologies emerge to address applications such as gene expression, it can get quite confusing to determine which approach is the best, whether one simply replaces the other, or do they work together to tell a more complete story? Here we try to demystify how to choose between real-time PCR (qPCR) and next-generation sequencing (NGS), or whether you really need to choose at all.
There are two primary approaches for whole transcriptome NGS: RNA-Seq and targeted transcriptome. RNA-Seq is recommended when one or more of the following are key to your research efforts:
You want to detect all cellular RNA: mRNA, miRNA, tRNA, etc.
You want to look at transcript isoform diversity/expression
Novel discovery is critical, i.e. you want to identify differentially expressed genes without a priori knowledge of which targets might be impacted by your future treatment
Targeted transcriptome approaches are similar in that novel discovery is possible, although the output will be limited to the most common mRNA transcripts, generally in the range of 20,000 targets spanning most of the known transcriptome.
Where does real-time PCR fit into these workflows? Data integrity is key to gene expression experiments, therefore it is common practice to use real-time PCR both upstream and downstream of NGS. This means that we don’t live in a “real-time PCR vs. NGS” world; rather the two technologies are complementary, working together to generate trustworthy results. Upstream of NGS, it is common to use TaqMan real-time PCR to check cDNA integrity prior to NGS.
Downstream of NGS, real-time PCR continues to be the go-to method for verification of results. In cases when verification is unnecessary, it is important to note that real-time PCR is the gold-standard technology for follow-up studies involving a targeted panel of transcripts discovered during the NGS screen.
The publications below feature Ion AmpliSeq NGS accompanied by TaqMan real-time PCR for either cDNA integrity checks or verification of results.
Read our technical note showing high concordance between TaqMan Gene Expression assays, Clariom D assays, and Ion AmpliSeq Transcriptome kits.
BIOZ biomedical research articles using both Ion AmpliSeq and TaqMan technologies
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Pre-treatment DNA methylome and transcriptome profiles correlate with melanoma response to anti-PD1 immunotherapy.
Cancer letters | 2025 Mar 13 | PubMed ID: 40089202 | Read Article
"...DNA quantification for Ion AmpliSeq™ Library Preparation was performed using TaqMan® RNase P Detection Reagents, following the manufacturer’s instruction (ThermoFisher). DNA samples were analysed usin...""DNA quantification for Ion AmpliSeq™ Library Preparation was performed using TaqMan® RNase P Detection Reagents, following the manufacturer’s instruction (ThermoFisher). DNA samples were analysed using Oncomine™ Tumor Mutation Load Assay (ThermoFisher, Melbourne, Australia). The resulting data were received in BAM file format for further analysis. Initial analysis was conducted using the Oncomine Tumour Mutation Load – w3.0 – DNA – Single Sample workflow on the Ion Reporter cloud server. All samples passed in-built QC (quality control) test, achieving ≥80 % uniformity in depth of reads across the whole genome. Mutation loads were then calculated as mutations per megabase (Mutations/MB) for each sample, with statistical significance at pvalue <0.05. We used Ion Reporter™ Software algorithm to calculate tumour mutational burden (TMB) using following formula:
TMB= ( SM × 106 )
Total exonic bases with sufficient coverage
• TMB = Tumour mutational burden • SM = Somatic mutations"More...
Skin-Protective Performance of Alternative Stratum Corneum Formed by a Pseudo-Ceramide-Containing Steroid Lamellar Cream.
"...Sequencing libraries were prepared using an Ion AmpliSeq Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific). Briefly, RNA was reverse- transcribed using a SuperScript VILO cDNA Synthes...""Sequencing libraries were prepared using an Ion AmpliSeq Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific). Briefly, RNA was reverse- transcribed using a SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). cDNA was ligated with Ion Xpress Barcode adaptors and purified using AMPure XP beads (Beckman Coulter, Brea, CA, USA). A quality check of the library was performed using a High Sensitivity D1000 ScreenTape on an Agilent 4200 TapeStation. Each library was quantified using an Ion Library
TaqMan Quantitation Kit (Thermo Fisher Scientific) and diluted to 100 pM prior to template preparation. Templates were prepared and loaded onto an Ion 540 chip in an Ion Chef System (Thermo Fisher Scientific). RNA- seq was performed using an Ion S5 XL System (Thermo Fisher Scientific)."More...
Retrospective nationwide survey of pediatric RDD in Japan: a high prevalence of mutations in the kinase pathway genes.
International journal of hematology | 2025 Mar 10 | PubMed ID: 40063331 | Read Article
"...eviously reported in histiocytoses (Supplemental Table 1). A DNA library was prepared using Ion AmpliSeq Library Kit Plus and DNA OCAv3 (Thermo Fisher Scientific) according to the manufacturer’s proto...""..ed using the TaqMan Copy Number Reference Assay and RNase P, respectively (Thermo Fisher Scientific, Waltham, MA, USA). Samples from which 20 ng or more DNA could be extracted were subjected to OncomineTM Comprehensive Assay v3 (OCAv3) analysis [14], which detects mutations (87 hotspot genes and 48 full coding sequence genes) and copy number changes (43 genes) in a total of 146 cancer-related genes, including mutations previously reported in histiocytoses (Supplemental Table 1). A DNA library was prepared using Ion AmpliSeq Library Kit Plus and DNA OCAv3 (Thermo Fisher Scientific) according to the manufacturer’s protocols. Briefly, DNA was amplified by the multiplex
polymerase chain reaction (PCR). The PCR products were ligated to Ion Xpress Barcode Adapters (Thermo Fisher Scientific) and purified using AMPure XP beads (Beckman Coulter, Brea, CA). The purified libraries were pooled and then sequenced using the Ion S5TM XL system and Ion 540 Chip (Thermo Fisher Scientific). DNA sequencing data were accessed through the Torrent Suite™ S.."More...
Quantitation of human mitochondrial DNA and whole mtGenomes sequencing of fingernail/hair shaft samples
Forensic Sciences Research | 2025 Mar 01 | 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...""..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...
Analysis of endometrial liquid‑based cytology samples to detect somatic mutations and classify ovarian cancer
"... storage, the L/S ratio was above 0.2, indicating that the samples could be analyzed by the Ion AmpliSeq Cancer Hotspot Panel v2. In Japan, the proportion of germline variants in ovarian cancer is app...""..of LBC specimens from patients with endometrial cancer has been reported to be useful in several studies, including the present study ( 23 – 25 ). As a preliminary experiment, ovarian cancer cell lines (SKOV, OVTOKO) were cultured in CelVerse TM as the cell preservation solution and stored refrigerated at 4°C, and the long assay/short assay ratio was calculated using the TaqMan ® Assay. Even after approximately 2 years of storage, the L/S ratio was above 0.2, indicating that the samples could be analyzed by the Ion AmpliSeq Cancer Hotspot Panel v2. In Japan, the proportion of germline variants in ovarian cancer is approximately 18%, and the major genes are BRCA1, BRCA2 , mismatch repair genes [MutL protein homolog ( MLH ) 1, MLH2, MLH6 , and postmeiotic segregation increased 2], RAD51 Paralog D, and ataxia telangiectasia mutated ( ATM ) ( 26 ). In contrast, TP53, PIC3CA, KRAS, ARID1A, CTNNB1, SMARCA4, BRCA1, BRCA2 , and ATR have been reported as driver genes in ovarian cancer ( 22 ). Of the 19 patients in the present study, one was di.."More...
Tracing Emergence of SARS-CoV-2 Variants: Insights from Comprehensive Assessment Using Reverse Transcription Polymerase Chain Reaction and Whole Genome Sequencing.
"...aqMan Omicron assay was used for variant typing for 122 samples. For mutation analysis, the Ion AmpliSeq SARS-CoV-2 research panel (Thermo Fisher Scientific, USA) was used during Periods A and B and e..."".. strategy was modified over time (Figure 1). During Period A, we used the TaqMan Delta assay for SARS-CoV-2 variant typing (Thermo Fisher Scientific, Waltham, MA, USA) and AccuPower RT-PCR (Bioneer Corporation, Daejeon, Republic of Korea) assays for all 219 samples. In Period B, we utilized the TaqMan Delta and TaqMan Omicron assays (Thermo Fisher Scientific, USA) for variant typing of 145 samples. In Period C, only the TaqMan Omicron assay was used for variant typing for 122 samples. For mutation analysis, the Ion AmpliSeq SARS-CoV-2 research panel (Thermo Fisher Scientific, USA) was used during Periods A and B and early Period C, but only for cases with discrepancies between assays or unidentified variants. In later Period C, the Illumina COVIDSeq assay (Illumina Inc., San Diego, CA, USA) was introduced. During Period D, dominated by the Omicron variant, we performed WGS using the Illumina COVIDSeq Assay to investigate Omicron sub-lineage patterns in 138 samples.
Microorganisms 2025, 13, x FOR PEER REVIEW 3 of 13
techniques used to .."More...
Circulating tumor cell plasticity determines breast cancer therapy resistance via neuregulin 1–HER3 signaling
Nature Cancer | 2025 Jan 03 | PubMed ID: 39753722 | Read Article
"...rrent mutations 51 . Library preparation and sequencing were performed using multiplex PCR-based Ion Torrent AmpliSeq (Thermo Fisher Scientific) and Ion S5XL technology, as previously described 52 . ...""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 BC 50 , covering the most recurrent mutations 51 . Library preparation and sequencing were performed using multiplex PCR-based Ion Torrent AmpliSeq (Thermo Fisher Scientific) and Ion S5XL technology, as previously described 52 . "More...
Type III interferons induce pyroptosis in gut epithelial cells and impair mucosal repair.
"...052
Aurum Total RNA Mini Kit Bio-Rad Cat#7326820
DNesay Blood & Tissue Kit QIAGEN Cat#69504 Ion AmpliSeq Transcriptome Mouse Gene Expression Panel, Chef-Ready Kit Thermo Fisher Scientific Cat#A36412
C...""..5
Cf594 Tyramide Amplification Kit with HRP Goat Anti-Mouse and CF Dye/Biotin Tyramide kit
Biotium Cat#33009
iScript cDNA synthesis kit Bio-Rad Cat#1708890
Power SYBR Green RNA-to-CT 1-Step Kit
Thermo Fisher Scientific Cat#4389986
Applied BiosystemsTaqManUniversal PCR Master Mix
Thermo Fisher Scientifc Cat#4304437
SuperScript VILO cDNA Synthesis Kit Invitrogen Cat#11754-05
Direct-zol RNA Miniprep Kit ZYMO RESEARCH Cat#R2052
Aurum Total RNA Mini Kit Bio-Rad Cat#7326820
DNesay Blood & Tissue Kit QIAGEN Cat#69504 Ion AmpliSeq Transcriptome Mouse Gene Expression Panel, Chef-Ready Kit Thermo Fisher Scientific Cat#A36412
Chromium Next GEM Single Cell 3’ GEM, Library & Gel Bead Kit v3.1, 4 rxns
10X Genomics Cat#PN-1000128
Chromium Next GEM Chip G Single Cell Kit 10X Genomics Cat#PN-1000127
NextSeq 2000 P3 Reagents (100 Cycles) Illumina Cat#20040559
Human Anti-Virus Response Panel (13-plex) with V-bottom Plate kit
Biolegend Cat#740390
Deposited data
Mouse bulk RNA sequencing datasubmitted to GEO database
This study GEO: GSE247149
IBD Patient.."More...
IDENTIFICATION OF CLINICALLY RELEVANT GENE VARIANTS IN COLON ADENOCARCINOMA SAMPLES OF UKRAINIAN PATIENTS USING A COMPREHENSIVE CANCER PANEL: A PILOT STUDY.
"...ermo Fisher, USA). 15 ng of gDNA was taken for library preparation. DNA was amplified using Ion AmpliSeq™ Comprehensive Cancer Panel Primer Pools and AmpliSeq HiFi mix (Thermo Fisher, USA). PCR pools ...""..thologies. The Helsinki Declaration was followed in conducting human sampling. Ethical approval was obtained from the Ethics Commis sion of Feofaniya Clinical Hospital of the State Management of Affairs Number 3 on May 24, 2024.
Colon cancer genotyping by Ion Torrent Gene Target Library preparation and NGS sequencing.
The genomic DNA (gDNA) was extracted from cancer samples using the GeneJET FFPE DNA Pu rification Kit (Thermo Fisher, USA). 15 ng of gDNA was taken for library preparation. DNA was amplified using Ion AmpliSeq™ Comprehensive Cancer Panel Primer Pools and AmpliSeq HiFi mix (Thermo Fisher, USA). PCR pools for each sample were combined and subjected to primer digestion with the FuPa reagent (Thermo Fisher, USA). Li braries were indexed using the Ion Xpress Barcode Adap ter Kit. After purification, the amplified li braries were quantified by qPCR with the TaqMan library quantification kit (Thermo Fisher, USA). All samples were diluted to a final concentration of 100 pM, then the amplicon libraries were pooled for emulsion PC.."More...
Anti-IL-4Rα aptamer reduces IL-4 signaling and formation of nasal polyps
"...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.
"...) 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.
"...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
"...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.
"... 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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993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 .."More...
Multi‐UniFocality (MUF), in contrast to multifocality, in thyroid lesions: Relation to lymphocytic thyroiditis
"...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...""..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
,
19
,
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 diagno.."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
"...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...
Real-time PCR and Illumina targeted amplicon RNA-Seq
Illumina targeted amplicon RNA-Seq is an NGS method for measuring 12-1,200 transcripts of interest. This range of targets overlaps with what is possible with real-time PCR (Figure 1). The choice between real-time PCR and Illumina targeted amplicon RNA-Seq should take into account these factors:
Historically, real-time PCR has been considered the gold standard for gene expression quantification. For this reason, real-time PCR instruments are ubiquitous, and the benchtop and data analysis workflows are familiar and straightforward. For an experiment involving 20 samples and 10 targets, with RNA samples and qPCR assays already in the freezer, all data can be generated in a few 96-well plates run across 1–2 days. The same experiment using Illumina targeted amplicon RNA-Seq would take longer, especially if an Illumina instrument was not easily accessible. Sending samples to a core facility or service provider would likely increase the sample-to-answer turnaround time.
Illumina targeted amplicon RNA-Seq will also cost more money. Targeted amplicon NGS is more expensive for experiments involving less than 20 targets.
Lastly, sensitivity and dynamic range are often cited as strengths of Illumina targeted amplicon RNA-Seq over real-time PCR given the absolute read-counting nature of NGS. It is worthy to note that in most experimental contexts, the sensitivity and dynamic range of real-time PCR are sufficient, and achieving top-level sensitivity in Illumina targeted amplicon RNA-Seq often requires sequencing to great depths, which drives up the cost of the experiment.
Click to enlarge Figure 1. Where each technology and format is most effective based on number of targets and samples.
Figure 2. Where real-time PCR outshines Illumina RNA-Seq
Yes, TaqMan Gene Expression assays are available for most exon-exon junctions for all genes and species in our predesigned assay collection. To select an assay that is variant-specific, first identify the NCBI RefSeq transcript accession number, and then use that accession number as your search term in the TaqMan Assay Search Tool. In the resulting list of assays, choose the assay that detects only that one variant of interest. If all available assays detect more than one RefSeq transcript, then use our Custom Assay Design Tool to attempt a variant-specific assay design.
Yes, TaqMan Array plates are 96- and 384-well plates containing 8–384 assays (pre-spotted and dried down, all you have to do is add cDNA and master mix). Based on the number of samples and targets needed, order just enough plates to build in technical and biological replication. TaqMan Array cards are 384-well microfluidic cards containing 12–384 assays with the added benefit of using less master mix than conventional plates and a super-easy benchtop workflow: pipet 384-wells in 2 minutes. TaqMan OpenArray plates are the highest-throughput option for qPCR offering the lowest price per data point.
It actually depends. The number of samples and targets possible in a single Illumina targeted amplicon RNA-Seq run depends on the Illumina platform and chemistry. Assuming the desired number of samples and targets can fit in a single Illumina targeted amplicon RNA-Seq run, then it will take two days from sample prep to data analysis. If you have to outsource your Illumina NGS experiment to a core facility or service provider, then it can take days or weeks to get your results. In contrast, the following real-time PCR experiments are possible in 1–3 days:
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