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View additional product information for Ion 530™ Chip Kit - FAQs (A27764)
47 product FAQs found
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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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
The Ion S5 GeneStudio Prime System has a maximum throughput/day of 50 Gb (2 Ion 550 Chips).
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The Ion S5 GeneStudio Prime System takes 8.5 hours to run an Ion 550 Chip.
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The Ion S5 GeneStudio Prime System takes 6.5 hours to run an Ion 540 Chip.
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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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Yes, the Ion GeneStudio S5 Prime System compatible with the Ion One Touch 2 System.
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Yes, the Ion GeneStudio S5 Prime System is compatible with the Ion Chef System.
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Yes, the Ion GeneStudio S5 Prime System has a separate server.
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The server storage size is 25 TB.
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The Ion GeneStudio S5 Plus System has a maximum throughput/day of 30 Gb (2 Ion 540 Chips).
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The Ion GeneStudio S5 Plus System takes 11.5 hours to run an Ion 550 Chip.
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The Ion GeneStudio S5 Plus System takes 10 hours to run an Ion 540 Chip.
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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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Yes, the Ion GeneStudio S5 Plus System compatible with the Ion One Touch 2 System.
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Yes, the Ion GeneStudio S5 Plus System compatible with the Ion Chef System.
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No, the server is contained inside the sequencer itself.
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The server storage size on the Ion GeneStudio S5 Plus System is 24 TB.
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Yes, the Ion GeneStudio S5 System compatible with the Ion One Touch 2 System.
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Yes, the Ion GeneStudio S5 System compatible with the Ion Chef System.
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The server storage size is 12 TB.
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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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No, the analysis of the first chip must complete before the sequencing run of the second chip begins.
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No, the Ion GeneStudio S5 System's analysis server is contained within the sequencer itself.
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The Ion GeneStudio S5 System takes 19 hours to run and analyze an Ion 540 Chip.
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The Ion GeneStudio S5 System has a maximum throughput/day of 15 Gb.
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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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The number of samples that can be multiplexed on the Ion 530 Chip will vary depending on whether automated or manual library preparation is used. If using automated library preparation, 32 somatic samples or 32 germline samples can be multiplexed on the Ion 530 Chip. If using manual library preparation, up to 96 germline samples can be multiplexed on the Ion 530 Chip. This is because of the available Ion Xpress Barcodes.
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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For 200 bp sequencing, it will yield 3-4 Gb. For 400 bp sequencing, it will yield 6-8 Gb.
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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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