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查看更多产品信息 Ion Library TaqMan™ Quantitation Kit - FAQs (4468802)
64 个常见问题解答
No, the Ion Library TaqMan Quantitation Kit for qPCR library quantification is unable to differentiate amplifiable primer-dimers from library fragments. We recommend assessing the library size distribution, including checking for the presence of adapter dimers, using the Bioanalyzer instrument. Libraries containing adapter dimers will have sharp peaks at ~70 bp for non-barcoded libraries or ~90 bp for barcoded libraries.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
The Ion AmpliSeq Microbiome Health Research Kit, Ion 550 bundle (Cat. No. A46497) consists of the Ion AmpliSeq Microbiome Health Research - 16S rRNA Gene Pool, the Ion AmpliSeq Microbiome Health Research - Target Species Pool, the Ion AmpliSeq Library Kit Plus, the IonCode Barcode Adapters, the Ion 550 Kit - Chef, and the Ion 550 Chip Kit for the rapid preparation of barcoded sample libraries from bacterial DNA. Sufficient reagents are provided to prepare uniquely barcoded DNA libraries from 384 research samples.
Note: The Ion Library TaqMan Quantitation Kit (Cat. No. 4468802) must be purchased separately.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
The Ion AmpliSeq Microbiome Health Research Kit, Library bundle (Cat. No. A46495) consists of the Ion AmpliSeq Microbiome Health Research - 16S rRNA Gene Pool, the Ion AmpliSeq Microbiome Health Research - Target Species Pool, the Ion AmpliSeq Library Kit Plus, and the IonCode Barcode Adapters for the rapid preparation of barcoded sample libraries from bacterial DNA. Sufficient reagents are provided to prepare uniquely barcoded DNA libraries from 48 research samples.
Note: The Ion Library TaqMan Quantitation Kit (Cat. No. 4468802) must be purchased separately.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
The Torrent Suite AmpliSeq_Microbiome_Health_ Analysis plugin and Ion Reporter Software workflows were developed and validated exclusively for the Ion AmpliSeq Microbiome Health Research Kit. Open source analysis tools may be available for your custom solutions.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
Customization solutions are available through AmpliSeq custom designs (Ampliseq.com), where you can customize your own assays for any species of your interest. For details, please contact your local Field Applications Scientist (FAS).
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
Several scientific publications have demonstrated the key role played by the gut microbial community in infection susceptibility and severity, including for SARS-CoV-2. The Ion AmpliSeq Microbiome Health Research Kit targets 73 species known to be associated with immune-related diseases, including a large number of known bacterial species with antiviral or immune-modulating properties.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
The Ion AmpliSeq Microbiome Health Research Kit is a targeted next-generation sequencing (NGS) assay that enables the profiling of the human gut microbiome by targeting eight of nine 16S hypervariable regions of 16S rRNA gene (16S rRNA Gene Pool) and allows for the accurate detection of 73 key bacterial species associated with human disease (Target Species Pool). The assay was validated on the Ion Chef and Ion GeneStudio systems and is available in different configurations, with only primers, primers and library prep reagents, or library prep, templating, and sequencing bundles. Check with your local Sales Rep for the best configuration for your experimental needs.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
A microbiome is a community of microorganisms living together in a particular habitat, for example the gut.
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- The overall library concentration may have been too low. We recommend renormalizing the individual libraries to 50 pM concentration and combining into a new library pool.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
- The overall library concentration may have been too high. We recommend diluting the library pool to 50 pM concentration.
- The individual library concentrations may have been too high. We recommend diluting the individual libraries to 50 pM concentration.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
- The individual libraries may have been normalized to different concentrations. We recommend renormalizing the individual libraries to 50 pM concentration and combining into a new library pool.
- The individual libraries may have been misquantified. We recommend requantifying the individual libraries, then normalizing the individual libraries to 50 pM concentration and combining into a new library pool.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
The overall library input into templating may have been low. We recommend renormalizing the individual libraries to 50 pM concentration and combining into a new library pool.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
The sample DNA may have been misquantified. We recommend requantifying the sample DNA using the Qubit dsDNA HS Assay Kit and Qubit Fluorometer.
Note: Library yield is not indicative of quality.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
Here are possible causes and solutions:
-The sample DNA may have been misquantified. We recommend requantifying the sample DNA using the Qubit dsDNA HS Assay Kit and Qubit Fluorometer.
-The sample quality may have been low. We recommend reextracting the sample DNA using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit or adding more sample DNA to the amplification reaction.
-The PCR, digestion, or ligation reactions may have been inefficient. We recommend ensuring proper dispensing and mixing of viscous components at each step.
-The AMPure XP beads may have been overdried. We recommend not drying the AMPure XP beads for more than 5 mins.
Note: Library yield is not indicative of quality.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
For the Ion 530 Chip, 8 libraries can be loaded. For the Ion 540 Chip, the number of libraries that can be loaded ranges from 8 to 32, depending on the size of the libraries. For the Ion 550 Chip, the number of libraries that can be loaded ranges from 12 to 48.
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The Ion AmpliSeq Mouse TCR Beta SR Assay, RNA is compatible with the Ion 530 Chip, Ion 540 Chip, and Ion 550 Chip.
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The Ion AmpliSeq Mouse TCR Beta SR Assay, RNA (Cat. No. A45489) consists of the Ion AmpliSeq Mouse TCR Beta-SR Panel, the Ion AmpliSeq Library Kit Plus (Cat. No. 4488990), and the Ion Torrent NGS Reverse Transcription Kit (Cat. No. A45003). Sufficient reagents are provided for the rapid preparation of 24 sample libraries.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
The sample RNA input can range from 25 ng to 1 µg.
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The Ion AmpliSeq Mouse TCR Beta SR Assay, RNA is compatible with a vast array of research sample types, including FFPE tissue, fresh-frozen (FF) tissue, sorted T cells, whole blood, periperal blood leukocytes (PBLs), and periperal blood mononuclear cells (PBMCs).
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The Ion AmpliSeq Mouse TCR Beta SR Assay, RNA (Cat. No. A45489) is a robust, targeted next-generation sequencing (NGS) assay designed to accurately identify and measure the clonal expansion of T lymphocytes by targeting the complementarity-determining region 3 (CDR3) of the T-cell receptor (TCR) gene locus from total RNA input.
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Up to 96 paired libraries (48 samples) can be combined and loaded onto a single Ion 550 Chip.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
The Ion AmpliSeq Microbiome Health Research Kit, Ion 540 bundle (Cat. No. A46496) consists of the Ion AmpliSeq Microbiome Health Research - 16S rRNA Gene Pool, the Ion AmpliSeq Microbiome Health Research - Target Species Pool, the Ion AmpliSeq Library Kit Plus, IonCode Barcode Adapters, the Ion 540 Kit - Chef, and the Ion 540 Chip Kit for the rapid preparation of barcoded sample libraries from bacterial DNA. Sufficient reagents are provided to prepare uniquely barcoded DNA libraries from 256 research samples.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
The minimum sample input is 1 ng/pool for the Ion AmpliSeq Microbiome Health Research - 16S rRNA Gene Pool and 10 ng/pool for the Ion AmpliSeq Microbiome Health Research - Target Species Pool.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
The Ion AmpliSeq Microbiome Health Research Kit was validated using DNA isolated from human stool samples to generate libraries for templating and sequencing on the Ion Chef and Ion GeneStudio systems. Different sample types and platform configurations may work but need to be validated by each lab. Contact your local Field Applications Scientist for assistance.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
The Ion AmpliSeq Microbiome Health Research Kit, Ion 540 bundle (Cat. No. A46496) is a targeted next-generation sequencing (NGS) assay that enables the profiling of the human gut microbiome by targeting eight of nine 16S hypervariable regions of 16S rRNA gene (16S rRNA Gene Pool ) and allows for the accurate detection of 73 key bacterial species associated with human disease (Target Species Pool). This bundle allows templating and sequencing with the Ion 540 Chip to be done on the Ion Chef and Ion GeneStudio systems.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
We recommend sequencing up to 5 DNA or RNA samples (4 samples and a non-template control (NTC)) on a single Ion 550 Chip.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
The Oncomine Comprehensive Assay Plus, manual library preparation (Cat. No. A46721) is designed to prepare barcoded sample libraries from DNA and RNA. The kit consists of Oncomine Comprehensive Assay Plus DNA (2-pool) (Part No. A45615), RNA Oncomine Comprehensive Assay v3M (2-pool) (Part No. A33637), and two kits of Ion AmpliSeq Library Kit Plus (Cat. No. 4488990). Sufficient reagents are provided to prepare libraries from 24 samples.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
10 ng of nucleic acid input per pool (20 ng of DNA and 20 ng of RNA) isolated from FFPE samples, including fine needle biopsies, is needed for the Oncomine Comprehensive Assay Plus, manual library preparation.
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The assay is optimized for use with formalin-fixed paraffin embedded (FFPE) samples.
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The Oncomine Comprehensive Assay Plus, manual library preparation (Cat. No. A46721) is a targeted, next-generation sequencing (NGS) assay that enables the detection of single nucleotide variants (SNVs), insertions and deletions (INDELs), copy number variations (CNVs), gene fusions, splice variants, tumor mutational burden (TMB), and microsatellite instability (MSI) across 500+ unique genes. This version of the assay is designed for manual library preparation, not for automated library preparation using the Ion Chef System.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
There are two analysis workflows available:
1. Oncomine TagSeq Lung v2 Liquid Biopsy - w2.0 - Single Sample: Detects and annotates low-frequency variants including SNPs/Indels (down to 0.1% limit of detection), Fusions, and CNVs from targeted nucleic acid libraries (DNA & RNA) from the Oncomine Lung Cell‑Free Total Nucleic Acid Research Assay. This is compatible with DNA & RNA purified from cell-free total nucleic acids.
2. Oncomine TagSeq Lung v2 Tumor - w2.0 - Single Sample: Detects and annotates low-frequency variants including SNPs/Indels (down to 0.5% limit of detection), fusions, and CNVs from targeted nucleic acid libraries (DNA & RNA) from the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay. Due to deamination events caused by the FFPE process, the minimum alternative allele frequency is set to 0.3%. This makes it compatible with DNA & RNA purified from FFPE tumor tissue as well as fresh frozen tumor tissue.
Yes, there is a specific BED file and Hotspot file for the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay. Please contact your local Field Application Specialist (FAS) or Clinical Application Consultant (CAC) to request the BED files.
Through the use of Tag Sequencing technology, low limits of detection (LOD) can be achieved for different variant types*:
- For SNVs/short indels, an LOD of 0.1% can be achieved with sensitivity of ˜90% and specificity of >99%
- For fusions & MET exon skipping, an LOD of 1% can be achieved with sensitivity of >90% and specificity of >99%
- For MET CNV target, detection as low as 1.2-fold amplification can be achieved with sensitivity of >90% and specificity of >99%
*Sensitivity and specificity for each variant type were determined using a collection of contrived positive samples and cfNA isolated from normal healthy donors.
No, this assay uses Tag Sequencing technology. Cell-free DNA (cfDNA) and cell-free RNA (cfRNA) are found at extremely low concentrations in the plasma fraction of whole blood. Because of this low prevalence, Taq Sequencing technology is utilized in this assay. The technology attaches unique molecular tags to the gene-specific primers. After amplification, the tagged molecules are grouped based on the tags. Groups containing the same mutant variant 80% of the time or greater will be called positive. Using the Tag technology, groups that contain random errors generated through the library construction/sequencing process are removed.
Use cell-free total nucleic acid (cfNA) extracted using a method optimized for cfNA isolation from plasma. We recommend the MagMAX Cell Free Total Nucleic Acid Isolation Kit (Cat. No. A36716). You can expect 5-50 ng of cfDNA and 5-100 pg of cfRNA from 10 mL blood research sample collected in a K2 EDTA blood collection tube (Cat. No. ??)
We recommend using the fluorescence-based Qubit dsDNA HS Assay Kit (Cat. No. Q32851) for quantification of cfNA and cfDNA samples. Spectrophotometric quantification methods are not recommended, because they are not reliable when the nucleic acid concentration is low. Use of these methods can lead to gross overestimation of the concentration of sample, under seeding of the target amplification reaction, and low library yields. There isn't a good absolute quantitation method for cfRNA.
We recommend using the Tag Sequencing BC Set 1-24 (Cat. No. A31830) and Tag Sequencing BC Set 25-48 (Cat. No. A31847) to generate barcoded libraries for multiplexed templating and sequencing.
We recommend sequencing the libraries on the Ion S5 or Ion S5 XL systems. The assay has been developed and verified for use on the the Ion 510 & Ion 520 & Ion 530 Kit - Chef, which requires Torrent Suite Software 5.4 or higher. Performance has been demonstrated using the Ion 540 Kit - Chef which requires Torrent Suite Software 5.2 or higher.
Here are the components of the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay:
- cfDNA Library PCR Master Mix
- Low TE Buffer
- Lung cfTNA Panel
- cfDNA Library Primer P1
- Tag Sequencing BC1
- SuperScript VILO Master Mix
It contains a single pool of multiplex PCR primers.
1 sample can be multiplexed on an Ion 318 chip, up to 6 samples can be multiplexed on an Ion 530 chip, and up to 24 samples can be multiplexed on an Ion 540 chip
The number of reads needed to detect single nucleotide variants (SNVs) for each library is ˜2.5 million. This determines hpw many samples can be multiplexed on a chip.
K2 EDTA blood collection tubes are preferred and can be purchasd from any major lab supplier. You can also use Cell-Free DNA BCT tubes from Streck (Cat. No. 218962).
For the best results, we recommend using plasma fraction from whole blood with minimal-to-low level of hemolysis. To prevent hemolysis during blood collection, please follow guidelines provided here (http://blog.fisherbioservices.com/avoiding-hemolysis-in-blood-sample-collection-and-processing blog). The Oncomine Lung Cell-Free Total RNA (cfNA) Research Assay is compatible with FFPE samples.
Note: Plasma samples with minimal-to-mild hemolysis are recommended to achieve minimal SNV false positives.
Yes, reverse transcription using the SuperScript VILO Master Mix is needed to convert cell-free RNA (cfRNA) into cDNA to enable structural variant detection (gene fusions and exon skipping).
The Oncomine Lung Cell-Free Total Nucleic Acid Research Assay contains sufficient reagents to prepare 8 libraries from cell-free total nucleic acid.
The input amount range is 1-50 ng and the recommended input amount is 20 ng. There is a high primer dimer peak when using <5 ng input. The higher input will shift the family size distribution (i.e., more reads will be needed to call out a variant). LOD (limit of detection) for SNV/small indels calls and fusion detection is more sensitive to the input amount whereas LOD for CNV calls is only slightly impacted by the input amount.
The Oncomine Lung Cell-Free Total Nucleic Acid Research Assay is part of a complete solution to detect lung tumor-derived cell-free DNA and RNA (cell-free total nucleic acid; cfNA) isolated from the plasma fraction of whole blood. This assay enables the analysis of:
- Hotspot genes (SNVs) and short indels: ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1, and TP53 (˜168 hotspots covered). These genes have been identified as frequently mutated in non-small cell lung cancer (NSCLC).
- Gene fusions: ALK, RET, ROS1
- MET exon 14 skipping
- Copy number gene (CNV): MET
We recommend using T Cell Leukemia (Jurkat) Total RNA (Cat. No. AM7858) as a control. T cell Leukemia (Jurkat) Total RNA is derived from a cell line consisting of a single T cell clonotype. Running the Oncomine TCR Beta-LR Assay on Jurkat Total RNA should enable detection of a single clonotype.
Unlike other AmpliSeq panels, there are no BED files and no reference is needed. All downstream analysis takes place in Ion Reporter Software.
We have tested the Ion Library Equalizer Kit with this kit for library quantitation and it does function, but manually balancing library input into the Ion Chef instrument is preferred.
Torrent Suite Software 5.6 and Ion Reporter Software 5.6 are needed to generate reports. In Torrent Suite Software 5.6, two Planned Run templates are provided for use with the Oncomine TCR Beta-LR Assay.
The Oncomine TCR Beta-LR Assay has been developed and verified for use on the Ion GeneStudio S5 Series System. The assay can be used with the Ion PGM System, however, this instrument configuration is not supported.
Here are the components of the Oncomine TCR Beta-LR Assay:
- Oncomine TCR Beta‑LR Panel
- Ion AmpliSeq Library Kit Plus
- Ion Select Barcode Adapters
The primer pool is at 5X concentration.
The Oncomine TCR Beta-LR Assay enables long-read RNA sequencing (up to 400 bp amplicon length) for complete characterization of the T cell receptor Beta (TCR Beta) chain, including all three complementarity-determining regions (CDR1, 2, and 3) of the variable gene. This enables allele-specific interrogation of TCR Beta to potentially predict drug-mediated adverse events.
The primers are provided as a single pool.
Up to 16 samples can be multiplexed on an Ion 530 chip, up to 4 samples can be multiplexed on an Ion 520 chip, and up to 4 samples can be multiplexed on an Ion 318 chip.
A variety of research sample types including fresh-frozen tissue, whole blood, and sorted T cells are compatible with the Oncomine TCR Beta-LR Assay.
T cell repertoire sequencing helps:
- To characterize diversity and study features of T lymphocytes in blood and infiltrated tumors
- To study variable gene polymorphisms implicated in autoimmune disease or immune-mediated adverse events
- To study the production and function of therapeutic or engineered T cells
This kit is good for 24 samples.
The Oncomine TCR Beta-LR Assay is an RNA-based next-generation sequencing (NGS) research assay that enables the characterization of the T cell receptor Beta (TCR Beta) sequences including all three complementarity-determining regions (CDR1, 2, and 3) of the variable gene. The assay measures T cell repertoire diversity as well as clonal expansion, and allows for identification of allele-specific polymorphisms.
If you are not using the Ion Library Equalizer Kit for library normalization, one of the following can be used (but the library should be at 100 pM):
- Ion Library TaqMan Quantitation Kit
- Qubit 3.0 Fluorometer or Qubit 2.0 Fluorometer
- Qubit dsDNA HS Assay Kit
- Agilent 2100 Bioanalyzer Instrument
- Agilent High Sensitivity DNA Kit
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
We recommend using the qPCR result, as quantification by qPCR is generally more accurate than quantification using the Bioanalyzer instrument.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
You can find library protocols, as well as other valuable information on NGS Sequencing, at our NGS Support Center (thermofisher.com/ngssupport). For all other protocols, please go here (http://www.thermofisher.com/manuals-protocols-search.html).
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.