Transitioning from TruSeq Custom Amplicon to AmpliSeq for Illumina

This resource provides step-by-step guidance through the process of transitioning from TruSeq Custom Amplicon to AmpliSeq for Illumina.

Initial Considerations

Transitioning a Current Custom Amplicon Panel

You can use DesignStudio to recreate a TruSeq Custom Amplicon panel as an AmpliSeq for Illumina panel. After the panel is designed and ordered with the Library Plus kit and AmpliSeq for Illumina index kit, the panel will be ready for validation runs.

Resources Supporting Panel Design

DesignStudio has built-in tooltips and online help to guide you through the custom panel design process. The AmpliSeq for Illumina Custom and Community Panels Support Page contains useful information including the Library Preparation User Guide, training videos, etc. For additional assistance, contact Illumina Technical Support or your Field Application Scientist.

Comparability of Coverage

After the panel design has completed in DesignStudio, a % in silico coverage will be provided. The actual empirical uniformity of coverage performance (% > 0.2x of the mean) will be within a few percentage points of the in silico coverage. For further assistance with optimizing coverage, contact your local field representative to engage with Concierge.

Compatible Sample Types

AmpliSeq for Illumina is compatible with DNA extracted from FFPE. This technology is capable of low input samples as low as 1 ng.

Accounting for Deamination Artifacts in FFPE Samples with Dual Strand Design

In general, the false positive variant call rate introduced by deamination of cytosines is very low, and very low frequency (< 1%) with the AmpliSeq for Illumina assay. If deamination is a major concern, UDG pretreatment of FFPE extracted DNA can be performed as recommended by the UDG enzyme manufacturer protocol.

Testing the Design

DesignStudio returns high confidence amplicon designs. Because each design is unique and sample input can vary, performance of the design needs to be tested empirically. In silico design coverage is a good predictor of actual panel performance.

Instrument Compatibility

These panels are ideal to run on the iSeq 100, MiniSeq, MiSeq, or NextSeq Series systems.

Workflow Considerations

Required Equipment

The thermal cyclers recommended for TruSeq Custom Amplicon Low Input and TruSeq Custom Amplicon v1.5 are not recommended for AmpliSeq for Illumina. Refer to the recommended list of thermal cyclers, compatible plates, and magnets in the Consumables and Equipment list.

Kit Components

AmpliSeq for Illumina requires a different set of reagent kits and indexes than the TruSeq Custom Amplicon kit. Refer to the Consumables and Equipment list for a full list of required components. Note the following differences relative to the requirements for TruSeq Custom Amplicon.

The following must be ordered separately:

  • Custom Panel
  • Library Plus kit
  • Index kit

You will need to purchase AMPure beads.


The ordering process is the same as TruSeq Custom Amplicon. Place orders through MyIllumina.

A reorder requires new synthesis of the custom panel and can be ordered from DesignStudio or through MyIllumina. The difference between initial order and reorder is that the design is already created. There is no difference in lead time between a reorder and a new custom panel order.

Sample QC Recommendations

Low-quality inputs using the protocol modifications indicated in the user guides have been successful. Commercially available or laboratory validated DNA extraction methods typically yield DNA that is compatible with the AmpliSeq for Illumina assay.

Panel Design

Designing Panels Using DesignStudio

TruSeq Custom Amplicon projects must be redesigned in DesignStudio. The designs can be exported from TruSeq Custom Amplicon to a *.bed file and then uploaded as an AmpliSeq for Illumina design input. See the DesignStudio Online Help for instructions.

Creating a Panel Design

When creating a panel, it is important to assess the overall coverage that it will deliver. A file containing the successfully targeted regions and a file containing design gaps will be available for review and identification of areas lacking coverage. You can design a panel in DesignStudio without committing to purchase.

DesignStudio provides coverage calculation for the overall panel design. You can also download gap files to identify areas lacking coverage. Access to the AmpliSeq for Illumina panel design tool is available from the Illumina website.

Library Preparation

Recommended Input

The recommended input amount is 10 ng per pool. The allowed range is 1–100 ng per pool.

AmpliSeq for Illumina works with low-quality DNA extracted from FFPE samples. For low-quality samples, or if sample quality is not known, three additional PCR cycles are recommended in the amplification target step. See the Adjustments to Thermal Cycler Program table in the Amplify DNA Targets section of the AmpliSeq for Illumina Custom and Community Panels Reference Guide.

Total Library Prep Time

The total library prep time for AmpliSeq for Illumina is 5–7 hours. The hands-on time is < 1.5 hours, not including library quantification, normalization, and pooling.

AmpliSeq for Illumina Index Adapter Sequences

The index adapters used in AmpliSeq for Illumina protocols are specifically designed for AmpliSeq for Illumina. Adapters have proprietary design and modifications. Nextera or TruSeq adapters are not compatible with this protocol.

See the AmpliSeq for Illumina Panels section of the Illumina Adapter Sequences Document for the adapter sequences and the AmpliSeq for Illumina Pooling Guidelines section of the Index Adapters Pooling Guide.

Library Normalization, Pooling, and Loading

AmpliSeq for Illumina libraries need to be normalized before pooling, then diluted to an optimal loading concentration. For guidance refer to the AmpliSeq for Illumina Custom and Community Panels Reference Guide.


Planning a Run

You can use Illumina Experiment Manager (IEM), which will also work with your previous TruSeq Custom Amplicon workflows. BaseSpace Sequence Hub Prep tab, and Local Run Manager will have AmpliSeq for Illumina have the new assays ready for planning your runs.

Options for setup
Run setup option Sequencing system Considerations Resources
IEM v1.15 or higher All Check system requirements before installing Download the installer from the IEM Software Downloads page
BaseSpace Sequence Hub Prep tab MiniSeq, NextSeq Series Register for a BaseSpace Sequence Hub account Access at
Local Run Manager off-instrument All Check system requirements before installing Download the installer from the Local Run Manager Software Downloads page
Recommended Read Length

A 2×151 bp paired-end read is recommended for 140–275 bp amplicon sizes. Up to 2×301 bp paired-end run on the MiSeq is recommended for 375 bp amplicon sizes.


The actual number of samples that can be pooled together per sequencing run depends on the number of amplicons and the desired depth of sequencing coverage. An online calculator is available to help with these calculations.

Data Analysis

DNA Analysis Pipeline

The recommended pipelines for DNA analysis are the DNA Amplicon App in BaseSpace and Local Run Manager DNA Amplicon module.

System Requirements for Local Run Manager

For system requirements and guidance on installation of Local Run Manager, refer to the Installation section of the Local Run Manager Reference Guide.

CNV Analysis

Several of the AmpliSeq for Illumina panels have been designed to perform CNV calling as part of the analysis. BaseSpace Sequence Hub contains two apps for CNV analysis.

  • Training - The training app helps establish what baseline looks like (no CNVs or normal CNVs). In general, no fewer than four biological samples are recommended to train the panel-specific baseline. For more detailed information, refer to the OncoCNV publication: Multi-factor data normalization enables the detection of copy number aberrations in amplicon sequencing data.
  • Calling - The calling app takes one or more BAM files from your samples and a corresponding baseline file (from the trainer app) as input and calculates CNV statistics.

CNV analysis is available exclusively in BaseSpace Sequence Hub.

Comparability of VCF Files

The VCF format is the same whether generated using TruSeq Custom Amplicon or AmpliSeq for Illumina. Illumina BaseSpace Variant Interpreter can be used to perform annotation, interpretation, and filtering analysis on DNA Amplicon analyses.

Reusing Manifest Files

AmpliSeq for Illumina uses a new manifest format, so TruSeq Custom Amplicon manifest files cannot be reused with AmpliSeq for Illumina. Custom-designed content will have corresponding manifests made available. Manifests for predesigned panels are available on the Illumina website.

Reference Datasets

The following table shows data analysis options. The test data sets are from AmpliSeq for Illumina Ready-to-Use panels that can be used to demo the analysis software.

Method Description Test Data Sets
DNA Amplicon App Cloud-based analysis solution used to perform alignment and variant calling. MiniSeq: Cancer Hotspot v2 Panel (NA12878 and Horizon Samples)
Local Run Manager DNA Amplicon Analysis Module Local analysis solution used to perform alignment and variant calling. Currently not available
OncoCNV Trainer The app uses input control samples and the corresponding panel bed file to train a baseline to be used by the OncoCNV caller app. OncoCNV trainer and caller demo
OncoCNV Caller The app detects copy number variants based on circular binary segmentation and statistical tests, using normalized amplicon data. OncoCNV trainer and caller demo

Additional Resources for Library Preparation

Additional Resources for Sequencing and Analysis