Ion AmpliSeq™ Transcriptome Human Gene Expression Kit, 384 Assays - FAQs

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.

The Ion AmpliSeq RNA ERCC Companion Panel provides enough reagents to perform 96 manual reactions or 48 automated reactions.

To view data from the Ion AmpliSeq RNA ERCC Companion Panel, Torrent Suite Software version 5.10 or later is required and Version 5.10.0.3 or later of the ERCC_Analysis plugin for Torrent Suite Software is required.

We do not recommend using the Ion AmpliSeq RNA ERCC Companion Panel with FFPE samples or with any panels besides human and mouse Ion AmpliSeq transcriptome gene expression panels.

We recommend adding 1 µL of the Ion Ampliseq RNA ERCC Companion Panel per manual target amplification reaction and 8 µL per primer pool for an automated target amplification reaction on the Ion Chef Instrument.

The Ion AmpliSeq ERCC Companion Panel is compatible with Ion AmpliSeq Transcriptome Human Gene Expression Kit and Ion AmpliSeq Transcriptome Mouse Gene Expression Kit (not recommended for use with FFPE samples or panels outside of Ion AmpliSeq Human or Mouse Transcriptome kits). With the addition of the Ion AmpliSeq RNA ERCC Companion panel to your Ion AmpliSeq RNA panel, dynamic range and sensitivity can be similarly evaluated in Ion AmpliSeq RNA libraries that are prepared from RNA samples containing the ERCC RNA Spike-In Mix

The Ion AmpliSeq RNA ERCC Companion Panel consists of 10 primer pairs targeting 10 transcripts from the ERCC RNA Spike-In Mix representing a 13log2 dynamic range. Chosen for general GC balance and amenability for linear amplification (TaqMan qPCR), the selected ERCC targets produce R2 > 0.9 under normal experimental conditions.

The Ion AmpliSeq RNA ERCC Companion Panel is compatible with both manual and chef-ready versions of human and mouse Ion AmpliSeq transcriptome gene expression kits:

- Ion AmpliSeq Transcriptome Human Gene Expression Kit (Cat. No. A26325, A26326, or A26327)
- Ion AmpliSeq Transcriptome Mouse Gene Expression Kit (Cat. No. A36553, A36554, or A36555)
- Ion AmpliSeq Transcriptome Human Gene Expression Panel, Chef-Ready Kit (Cat. No. A31446)
- Ion AmpliSeq Transcriptome Mouse Gene Expression Panel, Chef-Ready Kit (Cat. No. A36412)

The TAC needs expression data in .chp file format. The ampliseqRNA analysis Plugin automatically generates these .chp formatted files. In addition, the TAC needs to be configured for analysis of the Ion AmpliSeq Transcriptome Human Gene Expression data with these two files: the Transcripts Annotation file (https://s3.amazonaws.com/IonTorrent/usbupdate/TAC_4.0/hg19_AmpliSeq_Transcriptome_21K_v1.transcript.csv) and TAC Analysis Parameters file (https://s3.amazonaws.com/IonTorrent/usbupdate/TAC_4.0/hg19_AmpliSeq_Transcriptome_21K_v1.tac_array_config). Your local support specialist can provide the bed file for the panel.

You may find the application note here: http://tools.thermofisher.com/content/sfs/brochures/AmpliSeq-Transcriptome-app-note.pdf

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

We recommend multiplexing up to 8 samples with this kit when using the Ion Proton system, or the S5 system with the 540 Chip.

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

The Ion Xpress barcode kit is recommended for libraries with multiple samples.

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

The FuPa reagent is a proprietary reagent that is used to partially digest the primers and phosphorylate the amplicons for adapter ligation.

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

As a starting point, we recommend 5,000 reads per amplicon for an Ion AmpliSeq RNA library targeting from 1-200 genes. The actual sequencing depth required depends on the expression levels of the gene targets in your sample RNA so the sequencing depth can be scaled up or down if needed.

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

The Ion AmpliSeq Human Transcriptome Panel requires at least 10 ng of RNA input, including FFPE samples.

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

We recommend using MagMAX-96 Total RNA Isolation Kit (Cat. No. AM1830), PureLink RNA Mini kit (Cat. No. 12183018A), or PureLink RNA Micro Scale Kit (Cat. No. 12183016) for total RNA isolation from cells or tissues. For FFPE tissue, we recommend RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Cat. No. AM1975). For best results, treat the RNA with DNase prior to library generation.

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

The TAC currently does not provide pathway analysis for small panels such as the Oncomine Immune Response Research Assay.

Yes, the TAC tool is available for free. It can be downloaded from this link (https://downloads.thermofisher.com/TAC/TAC_4.0.2/TAC4x64.exe).

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.

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

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.