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View additional product information for Ion AmpliSeq™ Kit for Chef DL8 - FAQs (A29024)
31 product FAQs found
A DNA fragment library is constructed from whole genomic DNA and is commonly used for whole genome resequencing or de novo sequencing. Briefly, the whole genomic DNA is fragmented or sheared, ligated with Ion-specific adapter sequences, and then size-selected for the library fragments of the desired length.
Amplicon libraries are constructed from PCR-amplified DNA fragments and are used for targeted sequencing (e.g., investigating variants at known genomic locations). There are two types of amplicon libraries, short and long.
A short amplicon library contains DNA fragments (targets) with lengths that are compatible with the Ion template preparation kits without any further shearing or fragmentation during library preparation. Additionally, no size-selection step is required, as the amplicons are already within the desired size range.
A long amplicon library contains DNA fragments (targets) with lengths that are longer than those compatible with the Ion template kits and requires further shearing or fragmentation during library preparation. The library preparation protocol for long amplicons is similar to fragment libraries.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
When handling barcode adapters, be careful to avoid cross-contamination by changing gloves frequently and by carefully removing the plate seal.
- Pulse vortex the IonCode Barcode Adapters plate several times.
- Centrifuge the IonCode Barcode Adapters plate for 1 minute at 1000 x g to collect contents.
- Carefully remove, then discard the plate seal.
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The IonCode Barcode Adapters 1-96 Kit consists of 1 x 96-well plate, with 20 µL of each barcode per well. The plate consists of 960 reactions in total with 10 reactions for each of the 96 barcodes.
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The IonCode Barcode Adapters 1-96 Kit consists of 1 x 96-well plate, with 20 µL of each barcode per well. The barcodes are pre-diluted, ready-to-use. The 96-well plate format allows multichannel pipetting for more efficient work.
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The IonCode Barcode Adapters 1-96 Kit is shipped on dry ice and we recommend storing at -30 degrees C to -5 degrees C in an upright position.
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After the completion of a run, the Ion Chef Instrument holds the barcoded libraries in the tube loaded in Position D of the Reagents cartridge. To avoid fluid loss due to evaporation, remove and cap the tube of combined barcoded libraries as soon as possible after run completion. Do not leave the tube in the instrument longer than 24 hours after the start of the run. After 24 hours from the start of the run, the instrument chiller will stop actively cooling, and the sample will be held at 27 degrees C.
The tube in Position D of the Ion AmpliSeq Chef Reagents DL8 Cartridge will contain 700 µL of combined barcoded libraries at completion of the run. The libraries are at >>100 pM (total combined library concentration) and are ready to use in template preparation. One can store unused portions of combined libraries at 4 to 8 degrees C for up to 1 month. For longer-term storage, store at -30 to -10 degrees C.
Yes, fewer than 8 samples may be processed per run but keep in mind that a run consumes kit reagents for 8 samples regardless of the sample number.
If you are processing ≤5 samples, we recommend that you quantify your output combined library by qPCR to ensure that an optimal concentration is used in templating reactions.
If you are processing fewer than 8 samples, it is preferable to add replicates or positive control samples to the run. Otherwise, pipet 15 µL of nuclease-free water as non-template control into column 1 wells that do not contain a DNA sample.
The primer pools should be added to the Reagent Cartridge on the Ion Chef Instrument in positions A and B. Tubes in Positions C and D can be left empty. Uncap all 4 tubes in positions A, B, C, and D in the Ion AmpliSeq Chef Reagents DL8 cartridge.
Each reverse transcription reaction requires 10 ng of DNase-treated total RNA (≥1.43 ng/µL).
For each target amplification reaction, use 300-30,000 copies of DNA (10 ng of mammalian gDNA) from normal or FFPE tissue.
Reverse transcription needs to be performed manually in the IonCode 96-well PCR plate, using the SuperScript VILO cDNA Synthesis Kit. SuperScript reverse transcriptase enzyme is not supplied in the kit and must be purchased separately. If the RNA was prepared from FFPE tissue and not previously heat-treated, we recommend heating at 80 degrees C for 10 min, then cooling to room temperature.
The Oncomine Focus Assay, Chef-Ready Library contains the following components needed to prepare libraries for 32 samples:
- Oncomine Focus Assay, DNA Chef-Ready Panel (2X) - contains the DNA primer pool
- Oncomine Focus Assay, RNA Chef-Ready Panel (2X) - contains the RNA primer pool
- Ion AmpliSeq Kit for Chef Reagents DL8 cartridge - allows automation of the library prep on the Ion Chef Instrument
Note: The SuperScript transcriptase for cDNA synthesis is not included and must be purchased separately.
After completion of a run, the Ion Chef Instrument holds the barcoded libraries in the tube loaded in Position D of the Reagents cartridge. Please see schematic on Page 20 of the User Guide (https://tools.thermofisher.com/content/sfs/manuals/MAN0013432_Ion_AmpliSeq_Library_Prep_on_Ion_Chef_UG.pdf). The tube in Position D will contain 700 µL of combined barcoded libraries. The libraries are at »100 pM (total combined library concentration) and are ready to use in template preparation. To avoid fluid loss due to evaporation, remove and cap the tube of combined barcoded libraries as soon as possible after run completion. After 24 hrs from the start of the run, the instrument chiller will stop actively cooling, and the sample will be held at 27 degrees C. Do not leave the tube in the instrument longer than 24 hrs after the start of the run. You can store unused portions of combined libraries at 4-8 degrees C for up to 1 month. For longer-term storage, store at -30 degrees C to -10 degrees C.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
You can run 8 DNA or cDNA samples with custom or ready-to-use 1- or 2-pool Ion AmpliSeq primer panels. RNA samples must be manually reverse transcribed to cDNA with the SuperScript VILO cDNA Synthesis Kit (sold separately) prior to library construction on the Ion Chef Instrument.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
Yes, Ion AmpliSeq 5X panels require dilution to 2X before use on the Ion Chef Instrument. Add 180 µL Nuclease-free Water or Low TE to 120 µL 5X panel.
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The Ion AmpliSeq Kit for Chef DL8 contains sufficient reagents and consumables for performing 4 Ion Chef runs, with up to 8 Ion AmpliSeq libraries prepared per Ion Chef run, so that would be 32 libraries in total. The libraries are normalized using the equalizer kit to 100 pM and pooled together into one tube to go back on the Ion Chef System for template preparation.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
If you are processing 5 or fewer samples, we recommend that you quantify your output combined library by qPCR to ensure that an optimal concentration is used in templating reactions.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
The primer pools should be added to the Reagent Cartridge on the Ion Chef Instrument in positions A and B. Please see schematic on Page 20 of the User Guide (https://tools.thermofisher.com/content/sfs/manuals/MAN0013432_Ion_AmpliSeq_Library_Prep_on_Ion_Chef_UG.pdf). Tubes in Positions C and D can be left empty.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
For libraries >200 bp we recommend using standard rather than FAST instrument run mode on the thermocycler.
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Optional sample tracking on the Ion PGM Torrent Server allows you to automatically follow samples grouped in “Sample Sets” from library and template preparation to chip loading, sequencing, and data analysis.
Automated sample tracking from library preparation to template preparation and sequencing is supported only for libraries prepared from one Sample Set in one Ion AmpliSeq for Chef run. If libraries from multiple Ion AmpliSeq for Chef runs are super-pooled in a Library Sample Tube in an Ion Chef template run, you will need to enter sample information manually when setting up a Planned Run.
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They are in the PCR plate, column 6 (Rows A to H).
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Please add 10 ng of each of the 8 samples to each well in Column 1 (Rows A to H) of the PCR plate.
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Yes, fewer than 8 samples may be processed in a run, but keep in mind that a run consumes kit reagents for 8 samples regardless of the sample number.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
The Ion AmpliSeq Kit for Chef DL8 enables robust, automated Ion AmpliSeq library preparation from genomic DNA or RNA using the Ion Chef Instrument and 1- or 2-pool Ion AmpliSeq primer panels. The kit contains sufficient reagents and consumables to prepare up to 32 barcoded and equalized libraries that are ready for template preparation using either the Ion Chef System or the Ion OneTouch 2 System. Library preparation from RNA additionally requires the SuperScript VILO cDNA Synthesis Kit, ordered separately. The kit components are as follows:
- Ion AmpliSeq Chef Reagents DL8 (4 cartridges)
- Ion AmpliSeq Chef Solutions DL8 (4 cartridges)
- Ion AmpliSeq Chef Supplies DL8 (1 box with 4 inserts)
Per insert:
-- Ion AmpliSeq Tip Cartridge DL8
-- Frames PCR Foil Seal
-- Enrichment Cartridge
- IonCode 0101-0132 in 96-well PCR Plates (dried) (1 set of 4 plates):
- IonCode 0101-0108 in 96 Well PCR Plate (red)
- IonCode 0109-0116 in 96 Well PCR Plate (yellow)
- IonCode 0117-0124 in 96 Well PCR Plate (green)
- IonCode 0125-0132 in 96 Well PCR Plate (blue)
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No hardware upgrades are required to run the Ion AmpliSeq Kit for Chef DL8 on the Ion Chef Instrument.
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The total instrument run time is approximately 7 hours.
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If you've reviewed your design and are not satisfied with the results, please click on the “Not happy with this design? Let us help” link to have an Ion AmpliSeq team member contact you about additional options for your design.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
The ~70 bp or ~90 bp peak is likely standard or barcoded adapter dimers, respectively. Adapter dimers may form during the adapter ligation step and are usually removed during the size selection process. The adapter dimers will amplify on the Ion Torrent Ion Sphere particles during template preparation and decrease the overall throughput of usable sequencing reads; thus, we highly recommend removing the adapter dimers by performing an additional clean-up step prior to template preparation.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
In addition to input RNA quality and accurate quantification, the clean-up and size selection steps are critical to generating a successful RNA-Seq library.
- Be sure to mix the nucleic acid binding beads well before dispensing, and follow the workflow and incubation times as closely as possible.
- Use fresh ethanol and pre-wet pipette tips prior to transferring ethanol, as the volume is critical for size selection.
- Remove residual ethanol before elution using a small-volume pipette. Do not over-dry or under-dry the beads.
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Ion AmpliSeq technology offers simple and fast library construction for affordable targeted sequencing of specific human genes or genomic regions. Based on ultrahigh-multiplex PCR, Ion AmpliSeq technology requires as little as 10 ng of input DNA to target sets of genes, making sequencing of FFPE samples routine on Ion PGM Systems.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.