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View additional product information for Ion 540™ Kit-Chef (2 sequencing runs per initialization) - FAQs (A30011)
64 product FAQs found
Yes, Ion S5 Sequencing Reagents (Part No. A27768) is used on the instrument for both Ion 540 and Ion 550 Chip sequencing.
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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.
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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.
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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).
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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.
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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.
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A microbiome is a community of microorganisms living together in a particular habitat, for example the gut.
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- The instrument may have encountered an error. We recommend recording the error shown on the instrument display, then tapping OK. For further assistance, contact Technical Support at techsupport@thermofisher.com.
- A consumable may have been improperly loaded. We recommend ensuring that all consumables are correctly loaded. For instructions, see the user guide for your templating kit. For further assistance, contact Technical Support at techsupport@thermofisher.com.
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- An obstruction may be present on or around the door mechanism. We recommend removing the obstruction blocking the door, then resuming normal instrument operation.
- The instument may be experiencing a hardware or software error. We recommend contacting Technical Support at techsupport@thermofisher.com.C8
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Multiple causes are possible. We recommend powering off the instrument, waiting for 30 secs and then powering on the instrument. If you continue to experience the issue, contact Technical Support at techsupport@thermofisher.com.
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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.
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- 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.
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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.
Up to 96 paired libraries (48 samples) can be combined and loaded onto a single Ion 550 Chip.
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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.
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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.
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
No, the Ion 16S Metagenomics Kit utilizes 400 bp reads, so it is incompatible with the Ion 540 Kit - Chef, which only yields 200 bp reads.
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The Ion 540 Kit - Chef is compatible with Torrent Suite Software v5.0 and above.
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Two Ion 540 chips can be prepped and loaded in 13 hours, with only 15 minutes of hands on time.
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The Ion 540 Kit - Chef can yield up to 80 M reads.
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No, the Ion 540 Kit - Chef can only perform 200 bp sequencing runs.
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The Ion 540 Kit - Chef (Cat. No. A27759) contains reagents and consumables for performing eight 200 bp runs (1 run per initialization). The Ion 540 Kit - Chef (Cat. No. A30011) contains reagents and consumables for performing eight 200 bp runs (2 runs per initialization).
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The initialization is stable for 24 hours. Both runs, if performing two 20 0bp runs, must be started within 24 hours of the end of the initialization.
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The Ion 540 Kit - Chef (Cat. No. A27759) contains reagents and consumables for performing eight 200 bp runs (1 run per initialization). The Ion 540 Kit - Chef (Cat. No. A30011) contains reagents and consumables for eight 200 bp runs (2 runs per initialization).
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The shelf life of the Ion 540 Kit - Chef is 12 months from the date of shipment.
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The components of the Ion 540 Kit - Chef can be found here (https://tools.thermofisher.com/content/sfs/manuals/MAN0010848_Ion540_Chef_PI.pdf).
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The Ion S5/Ion S5 XL wash and cleaning bottles can be placed in regular plastic recycling bins. The sequencing reagent cartridge must be disposed of in the hazardous waste bin.
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No, you should not run the post-run clean after the first Ion S5/Ion S5 XL run. Make sure that you DESELECT the post-run clean option in the run setup for the first run. Failing to do so will lead to the instrument automatically performing the cleaning procedure, which will waste the reagents for the second run. RESELECT the post-run clean for the second run so that the instrument can perform the cleaning procedure.
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We recommend sequencing the second chip within 8 hours after the Ion Chef Instrument run has finished.
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It is possible to prepare just one chip on the Ion Chef Instrument per run, utilizing the Ion S5 Chip Balance, which is included in the Ion S5 Installation Kit. Please note that the Ion Chef Instrument will still use up reagents for two chips.
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Two Ion 520 or 530 chips can be prepped and loaded in 13 hours, with only 15 minutes of hands-on time.
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The Ion 520 & 530 Kit - Chef is compatible with Torrent Suite Software v5.0 and above.
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No, the Ion 520 & 530 Kit - Chef (Cat. No. A30010), which can perform 2 runs per initialization, can only perform 200 bp sequencing. Use the Ion 520 & 530 Kit - Chef (Cat. A27757) if you would like to perform 400 bp sequencing runs. Please note that you can only perform 1 run per initialization with this kit.
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The Ion 520 & 530 Kit - Chef (Cat. No. A27757) contains reagents and consumables for performing 8 runs, either 200 bp or 400 bp (1 run per initialization). The Ion 520 & 530 Kit - Chef (Cat. No. A30010) contains reagents and consumables for performing eight 200 bp runs (2 runs per initialization).
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Using the Ion 520 & 530 Kit - Chef with the Ion 520 Chip will yield up to 5 M reads. Using the Ion 520 & 530 Kit - Chef with the Ion 530 Chip will yield up to 20 M reads.
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Yes, you can perform templating runs with two different types of chips (Ion 510, 520, and 530). However, you cannot mix template sizes (200-base and 400-base template run lengths) in a single run. To perform templating runs with mixed chip types, you must update the Torrent Suite Software to version 5.6 or later.
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The initialization is stable for 24 hours. Both runs, if performing two 200 bp runs, must be started within 24 hours of the end of the initialization.
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The Ion 520 & 530 Kit - Chef (Cat. No. A27757) contains reagents and consumables for performing 8 runs, either 200 bp or 400 bp (1 run per initialization). The Ion 520 & 530 Kit - Chef (Cat. No. A30010) contains reagents and consumables for performing 8, 200 bp runs (2 runs per initialization).
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The shelf life of the Ion 520 & 530 Kit - Chef is 12 months from the date of shipment.
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The components of the Ion 520 & 530 Kit - Chef can be found here (https://tools.thermofisher.com/content/sfs/manuals/MAN0010847_Ion_520_530_Chef_PI.pdf).
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You can perform one or two runs per initialization depending on the kit you have:
- Ion 510 & Ion 520 & Ion 530 Kit - Chef (1 sequencing run per initialization), Cat. No. A34019)
- Ion 510 & Ion 520 & Ion 530 Kit - Chef (2 sequencing runs per initialization), Cat. No. A34461)
- Ion 540 Kit - Chef (1 sequencing run per initialization), Cat. No. A27759
- Ion 540 Kit - Chef (2 sequencing runs per initialization), Cat. No. A30011
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.