Ion AmpliSeq™ Library Kit Plus, 24 reactions - FAQs

Both kits are used for the same function (preparation of amplicon libraries using Ion AmpliSeq panels). The difference is that the Plus kit is a technical improvement in relation to the 2.0 kit. It has a different and optimized formulation that achieves slightly better results with difficult samples.

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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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Here are possible causes and solutions:
- The sample DNA or RNA may have been misquantified. We recommend requantifying the DNA or RNA. See the user guide for your library preparation method.
- More than 100 ng of sample DNA or RNA may have been used. We recommend decreasing the number of amplification cycles by adding less DNA or RNA.
- If you are using the Agilent 2100 Bioanalyzer instrument, the markers may have been misassigned. We recommend ensuring that the markers are correctly assigned.
- The library may have been contaminated by high molecular weight DNA. We recommend one of the following:
a) Removing less supernatant in the first-round (0.5X) purification and to ensure that the bead pellet is not disturbed.
b) Increasing the AMPur XP Reagent volume from 25 µL (0.5X) to 35 µL (0.7X) in the first-round purification.
- Inserts may have concatemerized during the ligation steps. We recommend reducing nucleic acid input, requantifying samples with a Qubit Fluorometer, or reducing the target amplification cycle number.

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- The sample DNA or RNA may have been misquantified. We recommend requantifying the DNA or RNA. See the user guide for your library preparation method.
- Target amplification may have been inhibited by residual ethanol in the sample DNA or RNA. We recommend incubating the tube uncapped in a hood for 1 hr or using a Speed-vac for 5 mins at room temperature to remove excess ethanol.
- Library amplification may have been inhibited by residual ethanol from the AMPure purification. We recommend carefully removing all drops of ethanol before library amplification, then centrifuging the plate, if needed.
- The sample DNA or RNA may have been of low quality. We recommend adding more DNA or RNA or increasing the number of amplification cycles.
- PCR, digestion, or ligation may have been inefficient. We recommend ensuring proper dispensing and mixing of viscous reagents at each step.
- The AMPure XP Beads may have been over-dried. We recommend drying the beads for 5 mins. Do not over dry the beads.
- The AMPure XP Beads may have inhibited library amplification. We recommend transferring the library from the beads before amplification.
- The qPCR cycling time may have been too short. We recommend using standard qPCR cycling conditions instead of fast cycling for library designs >175 bp.

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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.

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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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We recommend sequencing up to 5 DNA or RNA samples (4 samples and a non-template control (NTC)) on a single Ion 550 Chip.

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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.

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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.

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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.

Yes, the Ion AmpliSeq Library Kit Plus is an improved version of the Ion AmpliSeq Library Kit 2.0. However, the components of the Ion AmpliSeq Library Kit Plus differ from those of the Ion AmpliSeq Library Kit 2.0, and hence cannot be mixed and matched.

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The kit contains the following components:

- 5X Ion AmpliSeq HiFi Mix (red cap), 120 µL
- FuPa Reagent (brown cap), 48 µL
- Switch Solution (yellow cap), 96 µL
- DNA Ligase (blue cap), 48 µL
- 25X Library Amp Primers (pink cap), 48 µL
- 1X Library Amp Mix (black cap), 1.2 mL
- Low TE, 6 mL

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The kit provides enough reagents for manually preparing 24 libraries.

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We recommend storing at -30 to -10 degrees C except for Low TE which should be stored at 15 to 30 degrees C.

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The kit is available in the following reaction sizes:
- 24 reactions (Cat. No. 4488990)
- 96 reactions (Cat. No. A35907)
- 384 reactions (Cat. No. A38875)

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Here are the kit components:

- Ion AmpliSeq Library Kit Plus (Part No. 4488990), qty. 1
- Ion AmpliSeq Exome RDY Panel 4x2 (Part No. 4489840), 4 plates for 2 libraries each

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Here are the kit components:

- Ion AmpliSeq Exome RDY Panel 1x8 (1 plate for 8 libraries)
- Ion AmpliSeq Library Kit Plus (sufficient for preparing eight exome libraries)

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