Ion AmpliSeq™ Library Kit 2.0, 96 reactions - FAQs

The Ion AmpliSeq Designer (www.ampliseq.com) supports custom designs for targets in the human and mouse genomes. There are two design option sizes 125-175 bp, recommended for degraded DNA samples, and 125-275 bp, recommended for standard DNA input. Ion Ampliseq technology is a simple, fast, and affordable targeted sequencing strategy based on ultrahigh-multiplex PCR. The library preparation is completed in as little as 3.5 hours using 10 ng of DNA per PCR. To learn more about Ion Ampliseq technology, please review the Ion Ampliseq FAQs.

For other genomes (non-human or non-mouse) or applications requiring longer reads (up to 400-base reads), we recommend designing your own amplicons.

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

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 Library Equalizer Kit (Cat. No. 4482298) is recommended for use with Ion AmpliSeq libraries and provides an alternative to library quantification methods by using bead-based technology to normalize the final library concentration to ~100 pM.

The Ion Library Equalizer Kit is fast and cost-effective compared to traditional quantification methods; however, it may be not be the right choice for all users. Briefly, the library is amplified with the Ion Equalizer Primers, captured onto Equalizer Beads, and the normalized library is eluted from the beads using a specially formulated Equalizer Elution Buffer. The final library is normalized to ~100 pM, but there is no quality control information (e.g., measured concentration or size distribution) that can be obtained, which is possible if using the recommended library quantification kits: Ion Library Quantitation Kit (qPCR), Qubit dsDNA HS Assay Kit (Qubit 2.0 Fluorometer), or Agilent High Sensitivity DNA Kit (Agilent Bioanalyzer instrument).

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

For template preparation, Ion AmpliSeq libraries are diluted to 100 pM, and the volume required for template preparation will vary depending on the template preparation kit used. Please see the Ion AmpliSeq Library Preparation User Guide for details regarding library dilutions and input into template preparation.

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

Long-term storage of diluted libraries is not recommended and can result in decreased/suboptimal performance due to the adherence of DNA to the tube. In general, it is best to make fresh dilutions from the library stock for template preparation as needed. Libraries diluted for template preparation may be stored in a sealed plate or 0.2 mL PCR tube at 4-8 degrees C for up to 48 hours.

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

A properly stored library (amplified or unamplified) should be stable for at least 1 year at -20 degrees C in a non frost-free freezer. We highly recommend storing the concentrated library in low TE, in a low-bind tube, and placing the tube on the freezer shelf (not door) to minimize temperature fluctuations. To further minimize freeze-thaw cycles, it may also be worthwhile to store multiple aliquots of a single library.

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

If your library concentration is greater than 20 nM, it is best to re-amplify your targets with less input DNA or reduce the number of target amplification cycles. Over-amplification can result in uneven coverage of amplicons and compromised uniformity.

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

After library amplification, typical concentrations are approximately 300-1500 ng/mL or 1000-5000 pM, as measured using the Qubit dsDNA HS Assay Kit or Agilent High Sensitivity DNA Kit, respectively.

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

After the initial target amplification, starting from 10 ng of DNA and amplifying 16 PCR cycles, the theoretical yield is 1x1010 molecules. The concentration of the amplified material will be too low to run on the Agilent 2100 Bioanalyzer instrument, even using an Agilent High Sensitivity DNA Kit.

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

One of the most common failure modes for Ion AmpliSeq library preparation is improper quantification of the DNA sample going into the target amplification. We recommend using the TaqMan RNase P Detection Reagents Kit (Cat. No. 4316831), or the Qubit dsDNA HS Assay Kit (Cat. No. Q32851 or Q32854). While the Qubit dsDNA HS Assay Kit is sufficient for high quality DNA (e.g., cell culture isolates, etc.), the TaqMan RNase P Detection Assay will be superior for quantifying amplifiable material in potentially degraded DNA (e.g., DNA derived from FFPE samples).

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

The Coriell Institute for Medical Research (http://ccr.coriell.org) is good source for high quality, ready-to-use DNA that has already been isolated and quantitated.

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

The recommended minimum coverage for germline mutation detection is ~30X, while the recommended minimum coverage for somatic mutation detection is ~500X. Since there will be a distribution of coverage across the amplicons in the library, we recommend targeting an average coverage ~5 times the recommended minimum coverage (150x or 2500x, respectively) to ensure >95% of the bases are covered at the minimum value. In some cases, more or less coverage may be required to achieve the desired variant detection levels.

With Torrent Suite Software v 3.6.2, we support both germline and somatic mutation detection on the Ion PGM and Ion Proton Systems. For the Ion PGM System variant detection down to 5% frequency and Ion AmpliSeq 125-175 bp and up to 275 bp designs are supported. For the Ion Proton System variant detection down to 10% frequency and Ion AmpliSeq 125-175 bp designs are supported.

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

Ion Ampliseq technology is a simple, fast, and affordable targeted sequencing strategy based on ultrahigh-multiplex PCR. The library preparation is completed in as little as 3.5 hours using 10 ng of DNA or RNA per PCR. DNA or RNA from a variety of sources—including formalin-fixed, paraffin-embedded (FFPE) tissue—can be used as the starting material.

For DNA input library preparation, you will need the Ion Ampliseq Library Kit 2.0, provided in 8, 96, or 384 reaction formats (Cat. Nos. 4475345, 4480441, or 4480442, respectively) in combination with one of the following Ion Ampliseq panels;
Ion Ampliseq DNA Ready-to-Use Panels:
- Ion AmpliSeq Cancer Hotspot Panel v2 (Cat. No. 4475346)
- Ion AmpliSeq Cancer Primer Pool (Cat No. 4471262)
- Ion AmpliSeq Comprehensive Cancer Panel (Cat. No. 4477685)
- Ion AmpliSeq Inherited Disease Panel (Cat. No. 4477686)
Ion Ampliseq Community Panels (ordered on Ampliseq.com):
- Ion AmpliSeq BRCA1 and BRCA2 Panel
- Ion AmpliSeq Colon and Lung Cancer Panel
Ion Ampliseq Custom DNA Panels (designed/ordered on Ampliseq.com)

For DNA input library preparation, we also offer the Ion Ampliseq Exome Kit (Cat. No. 4487084) as a complete, stand-alone kit.

For RNA input library preparation, you will need the Ion Ampliseq RNA Library Kit, provided in 8, 96, or 384 reaction formats (Cat Nos. 4482335, 4482340, or 4482752, respectively), in combination with one of the following Ion Ampliseq panels;
Ion Ampliseq RNA Ready-to-Use Panels;
- Ion AmpliSeq RNA Apoptosis Panel (Cat. No. 4482571)
- Ion AmpliSeq RNA Cancer Panel (Cat. No. 4482572)
Ion Ampliseq Custom DNA/RNA Panels (designed/ordered on Ampliseq.com)

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

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.

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

- 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.

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

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.