Targeted Resequencing | TruSeq Custom Amplicon

TruSeq Custom Amplicon

TruSeq Custom Amplicon Workflow

The TruSeq Custom Amplicon Assay provides the fastest and easiest multiplexed amplicon assay optimized for MiSeq. It is a highly targeted approach that enables a wide range of applications for discovering, validating, and screening genetic variants related to specific study objectives. Currently, the assay allows for the sequencing of up to 1,536 custom amplicons from up to 96 samples per MiSeq run. The assay also includes unique base calling algorithms automated for MiSeq's on-board data analysis and variant calling.

The TruSeq Custom Amplicon Assay begins with the design of custom amplicons to target regions of interest. One pair of oligos is designed for each amplicon. Hybridization of these oligos to unfragmented genomic DNA occurs in a 96-well plate, followed by extension and ligation to form DNA templates consisting of the regions of interest flanked by universal primer sequences, one upstream and one downstream of the genomic region of interest. Using indexed primers supplied with the kit, index genomic targets are then PCR amplified, pooled into a single tube, and sequenced on the MiSeq System. The data can be viewed using the Illumina Amplicon Viewer and, when runs are completed on a MiSeq, Illumina MiSeq Reporter.

For more information, see the TruSeq Custom Amplicon data sheet on Illumina.com.

DesignStudio Workflow For TruSeq Custom Amplicon

You can design and order TruSeq Custom Amplicon panels online using DesignStudio. DesignStudio is a free relatively simple interface for custom probe design, providing dynamic feedback to select desired amplicons and estimate total project pricing. After logging on to your personalized account, you can create a new TruSeq Custom Amplicon project and choose several project-level attributes, such as reference genome to design against and desired amplicon size. Once created, you can easily add genomic targets to your project by gene name, genomic coordinates or batch file upload. Amplicon designs are generated automatically using an algorithm that considers a range of factors, such as GC content, sequence similarity, presence of repetitive sequences, and possible underlying variants (SNPs). Once your design is complete, you can view candidate amplicons covering your regions of interest as well as their design scores. Please note that DesignStudio returns only the candidate amplicons that we believe will work well in the multiplex TruSeq Custom Amplicon assay. The amplicon scores presented are an estimate of the relative performance of a particular amplicon compared to all others in the pool. You can perform filtering with user-defined tags and amplicons can be added to or removed from the design. After visualization and QC, the custom amplicon set is added to the final design, and can then be ordered along with the recommended sequencing consumables.

How DesignStudio Works For TruSeq Custom Amplicon

You will first select amplicon length when entering project basic information. Design Studio uses this length to determine probe spacing within targets.

Advancing beyond the BASIC INFORMATION page, you will then choose target regions for the design. Regions are defined by genomic coordinates or gene name. These targets may range in size from 1bp - 24kb, with a cumulative maximum of 650kb per project. Design Studio uses the target coordinates and flanking sequence, equivalent to the amplicon length set by the user, as the range for designing probes. Consequently, two regions that are closer than one amplicon length will be merged and targeted as one contiguous region. This helps to reduce the number of probes designed for a project and limit probe-probe interactions. Merged targets will display a target proximity warning (TP), and a dialog window will request confirmation to proceed prior to the start of the design process. If you want individual targets, ensure that the targets with TP flags are greater than one amplicon length away from other targets.

Finally, you will submit the project for design. DesignStudio first checks targeted regions for known variants, GC content, and repetitive regions that can interfere with the creation of acceptable probes. Candidate probes are then generated to “tile” the targeted regions. Selected probes are checked against one another for cross-binding, overlap, and poor specificity to the target of interest. Probes that do not meet specifications are not used. Design Studio will attempt to redesign these regions with new candidates to cover any failed targets. DesignStudio then selects the final probe pool and assigns a design score to targets before they are returned to the user.

The result of this process is a TruSeq Custom Amplicon project that contains a pool of oligos that has been tested to ensure there are no anticipated probe-probe interactions or excessive off target hits. The design scores are based on the unique pool of targets and their interactions.

New targets cannot be added to an existing designed panel without going through the design process again. This ensures that completed projects have undergone Illumina algorithms to eliminate undesired probe interactions.