Ion 540™ Chip Kit, 8 Chips - FAQs

View additional product information for Ion 540™ Chip Kit - FAQs (A27766, A27765)

70 product FAQs found

How many samples can I multiplex on a single chip for sequencing on the Ion PGM or Proton System?

The number of samples that can be multiplexed in a single sequencing run depends on the capacity of the chip, the size of the library, and the required coverage. A table of approximate capacities based on the size of the library and chip can be found in the Ion Ampliseq Preparation User Guide (https://tools.thermofisher.com/content/sfs/manuals/MAN0006735_AmpliSeq_DNA_RNA_LibPrep_UG.pdf) within the section “Strategies for combining Ion Ampliseq libraries.”

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What is included in the Ion AmpliSeq Microbiome Health Research Kit, Ion 550 bundle?

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.

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What is included in the Ion AmpliSeq Microbiome Health Research Kit, Library bundle?

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.

Can I use the AmpliSeq_Microbiome_Health_ Analysis plugin for Torrent Suite Software and Ion Reporter Software workflows for my custom 16S or target species AmpliSeq panel?

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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Can I customize the list of target species for my microbiome diversity research project?

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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Can the Ion AmpliSeq Microbiome Health Research Kit be used to investigate the human response to SARS-CoV-2 infection?

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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What is the Ion AmpliSeq Microbiome Health Research Kit?

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.

What is a microbiome?

A microbiome is a community of microorganisms living together in a particular habitat, for example the gut.

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With the Ion AmpliSeq Microbiome Health Research Kit, I am seeing a high percentage (>15%) of low quality ISPs. Why is this?

- 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.

With the Ion AmpliSeq Microbiome Health Research Kit, I am seeing a high percentage (>40%) of poyclonal ISPs. Why is this?

- 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.

With the Ion AmpliSeq Microbiome Health Research Kit, I am seeing that the number of total reads per library is imbalanced. Why is this?

- 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.

With the Ion AmpliSeq Microbiome Health Research Kit, I am seeing a low number of total reads for the chip run. Why is this?

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.

Why is my library concentration high with the Ion AmpliSeq Microbiome Health Research Kit?

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.

Why is my library concentration low with the Ion AmpliSeq Microbiome Health Research Kit?

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.

How many samples from the Ion AmpliSeq Microbiome Health Research Kit, Ion 550 bundle can I run on the Ion 550 Chip?

Up to 96 paired libraries (48 samples) can be combined and loaded onto a single Ion 550 Chip.

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What is included in the Ion AmpliSeq Microbiome Health Research Kit, Ion 540 bundle?

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.

How much of each sample is needed for the Ion AmpliSeq Microbiome Health Research Kit?

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.

What types of sample is the Ion AmpliSeq Microbiome Health Research Kit compatible with?

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.

What is the Ion AmpliSeq Microbiome Health Research Kit, Ion 540 bundle?

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.

Can I use Ion 540 Chip Kit with Ion AmpliSeq Primary Immune Deficiency Research Panel v2? I am worried about the average amplicon length of 243 bp.

Yes, can use Ion 540 chips with an amplicon length of 243 bp. The 200 bp protocol, which is available for all chip sizes including Ion 540 and 550, can be used for amplicons up to 275 bp. Instead, the 400 bp protocol can be used only with Ion 510, 520, and 530 chips with amplicons lengths ranging between 125 and 375 bps.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

For the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay, what are the analysis workflows in Ion Reporter Software?

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.

Are there any BED files for the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay?

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.

What is the LOD (limit of detection) for the Oncomine Lung Cell-Free Total Nucleic Acid (cfNA) Research Assay?

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.

Does the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay use AmpliSeq technology?

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.

Which kit should I use to isolate cell-free total nucleic acid (cfNA) for the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay?

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. ??)

With the Oncomine Lung Cell-Free Total Nucleic Acid (cfNA) Research Assay, which method do you recommend for quantitating cfNA?

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.

With the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay, which kit should I use to generate barcoded libraries?

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.

Can I run the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay on the Ion PGM System?

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.

What are the components of the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay?

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

How many primer pools does the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay kit contain?

It contains a single pool of multiplex PCR primers.

How many Oncomine Lung Cell-Free Total Nucleic Acid Research Assay samples can I multiplex on a chip?

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.

Which blood collection tubes do you recommend using with the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay?

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).

Which research samples are compatible with the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay?

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.

For the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay, do I have to reverse transcribe the cell-free RNA (cfRNA)?

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).

How many samples can I prepare with the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay?

The Oncomine Lung Cell-Free Total Nucleic Acid Research Assay contains sufficient reagents to prepare 8 libraries from cell-free total nucleic acid.

What is the recommended input amount of TNA (total nucleic acid) for the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay?

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.

What does the Oncomine Lung Cell-Free Total Nucleic Acid Research Assay measure?

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

What is the maximum throughput/day for the Ion S5 GeneStudio Prime System?

The Ion S5 GeneStudio Prime System has a maximum throughput/day of 50 Gb (2 Ion 550 Chips).

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

How long does it take to run an Ion 550 Chip on the Ion S5 GeneStudio Prime System?

The Ion S5 GeneStudio Prime System takes 8.5 hours to run an Ion 550 Chip.

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How long does it take to run an Ion 540 Chip on the Ion S5 GeneStudio Prime System?

The Ion S5 GeneStudio Prime System takes 6.5 hours to run an Ion 540 Chip.

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What chips are compatible with the Ion GeneStudio S5 Prime System?

Ion 510 Chip, Ion 520 Chip, Ion 530 Chip, Ion 540 Chip, and Ion 550 Chip are compatible with the Ion GeneStudio S5 Prime System.

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Is the Ion GeneStudio S5 Prime System compatible with the Ion One Touch 2 System?

Yes, the Ion GeneStudio S5 Prime System compatible with the Ion One Touch 2 System.

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Is the Ion GeneStudio S5 Prime System compatible with the Ion Chef System?

Yes, the Ion GeneStudio S5 Prime System is compatible with the Ion Chef System.

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Does the Ion GeneStudio S5 Prime System have a separate server?

Yes, the Ion GeneStudio S5 Prime System has a separate server.

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What is the server storage size on the Ion GeneStudio S5 Prime System?

The server storage size is 25 TB.

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What is the maximum throughput/day for the Ion GeneStudio S5 Plus System?

The Ion GeneStudio S5 Plus System has a maximum throughput/day of 30 Gb (2 Ion 540 Chips).

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How long does it take to run an Ion 550 Chip on the Ion GeneStudio S5 Plus System?

The Ion GeneStudio S5 Plus System takes 11.5 hours to run an Ion 550 Chip.

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How long does it take to run an Ion 540 Chip on the Ion GeneStudio S5 Plus System?

The Ion GeneStudio S5 Plus System takes 10 hours to run an Ion 540 Chip.

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What chips are compatible with the Ion GeneStudio S5 Plus System?

Ion 510 Chip, Ion 520 Chip, Ion 530 Chip, Ion 540 Chip, and Ion 550 Chip are compatible with the Ion GeneStudio S5 Plus System.

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Is the Ion GeneStudio S5 Plus System compatible with the Ion One Touch 2 System?

Yes, the Ion GeneStudio S5 Plus System compatible with the Ion One Touch 2 System.

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Is the Ion GeneStudio S5 Plus System compatible with the Ion Chef System?

Yes, the Ion GeneStudio S5 Plus System compatible with the Ion Chef System.

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Does the Ion GeneStudio S5 Plus System have a separate server?

No, the server is contained inside the sequencer itself.

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What is the server storage size on the Ion GeneStudio S5 Plus System?

The server storage size on the Ion GeneStudio S5 Plus System is 24 TB.

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Is the Ion GeneStudio S5 System compatible with the Ion One Touch 2 System?

Yes, the Ion GeneStudio S5 System compatible with the Ion One Touch 2 System.

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Is the Ion GeneStudio S5 System compatible with the Ion Chef System?

Yes, the Ion GeneStudio S5 System compatible with the Ion Chef System.

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What is the server storage size on the Ion GeneStudio S5 System?

The server storage size is 12 TB.

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How long does analysis take on the Ion GeneStudio S5 System?

Please see the following table (https://www.thermofisher.com/us/en/home/life-science/sequencing/next-generation-sequencing/ion-torrent-next-generation-sequencing-workflow/ion-torrent-next-generation-sequencing-run-sequence/ion-s5-ngs-targeted-sequencing/ion-s5-specifications.html).

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Can the Ion GeneStudio S5 System run two chips back-to-back?

No, the analysis of the first chip must complete before the sequencing run of the second chip begins.

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Does the Ion GeneStudio S5 System have a separate analysis server?

No, the Ion GeneStudio S5 System's analysis server is contained within the sequencer itself.

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How long does it take to run an Ion 540 Chip on the Ion GeneStudio S5 System?

The Ion GeneStudio S5 System takes 19 hours to run and analyze an Ion 540 Chip.

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What is the maximum throughput/day for the Ion GeneStudio S5 System?

The Ion GeneStudio S5 System has a maximum throughput/day of 15 Gb.

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What chips are compatible with the Ion GeneStudio S5 System?

Ion 510 Chip, Ion 520 Chip, Ion 530 Chip, and Ion 540 Chip are compatible with the Ion GeneStudio S5 System.

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What are the shipping and storage conditions for the Ion 540 chip?

The Ion 540 chip is shipped at room temperature and should be stored at room temperature.

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Can I reuse the Ion 550 Chef Reagents and Ion 550 Chef Solutions cartridges for running a second chip separately from the first chip?

The Ion 550 Chef Reagents and Ion 550 Chef Solutions cartridges can be reused in a second single-chip templating run if the second run is performed within eight days of the first run.

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Which instrument platforms can I use with the Ion 540 chip?

The Ion 540 chip is compatible with the Ion GeneStudio S5/S5 Plus/Prime and Ion S5/S5 XL instruments.

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Can I perform templating with either the Ion OneTouch 2 System or the Ion Chef System when using the Ion 540 chip for sequencing?

The Ion 540 chip workflow is supported with both the Ion OneTouch 2 System and the Ion Chef System for templating.

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What is the largest length I can sequence with an Ion 540 chip?

Up to 200 bp libraries (500 flows) are supported on the Ion 540 chip.

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How many reads can I expect with an Ion 540 chip?

You may expect 60-80 million reads with an Ion 540 chip.

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What is the throughput of the Ion 540 Chip?

For 200 bp sequencing, it will yield 10-15 Gb. 400 bp sequencing is not supported on the Ion 540 Chip.

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What Ion PGM chips are available? How much sequencing data can I expect from each chip type?

The Ion PGM System supports the Ion 3 series chips, including the Ion Torrent Ion 314 Chip Kit v2 (Cat. No. 4482261), Ion Torrent Ion 316 Chip Kit v2 (Cat. Nos. 4483188 and 4483324), and Ion Torrent Ion 318 Chip Kit v2 (Cat. Nos. 4484354 and 4484355). The Ion 314 Chip Kit v2 is available in an 8-pack; the Ion 316 Chip Kit v2 and Ion 318 Chip Kit v2 are available in 4-packs and 8-packs.

For more details, please see the Ion PGM System Specification Sheet (https://tools.thermofisher.com/content/sfs/brochures/PGM-Specification-Sheet.pdf).

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