Targeted sequencing of your most important genes
Tailored to meet your research needs, custom amplicon panels offer a completely curated targeted sequencing workflow to interrogate genomic targets relevant to you.
xGen NGS—made to discover.
This technology enables design of custom panels for targeted sequencing, offering a completely curated, targeted NGS workflow to rapidly interrogate genomic targets relevant to your research.
This technology enables low-frequency variant identification ≤1% through the incorporation of molecular identifiers (MIDs or UMIs), to promote improved sensitivity and specificity by removing false positive errors introduced during PCR and sequencing.
To submit a design request, use the form and template.Design Request
Any xGen Custom Amplicon Panel (HS included) will require a custom part number to be generated at point of order.
For customers who wish to purchase the legacy rhAmpSeq™, please contact us today.
xGen Custom Amplicon Panels are custom-designed primer pools to create multiple overlapping amplicons for specific genetic targets in research studies (Figure 1). Panels can multiplex hundreds to thousands of targets in a single tube using a simple 2-hour workflow (Figure 2). Compatible with cell-free DNA and FFPE (formalin-fixed paraffin embedded) research samples starting with as little as 10 ng input or pathogen RNA/DNA in the picogram range. For a listing of the specifications for custom panels, see Table 1. If multiple libraries are being examined at the same time, the final NGS library can be normalized using the IDT proprietary xGen Normalase reagent.
The cost-effective and flexible custom design means the panel can be used for research applications such as:
Figure 1. xGen Custom Amplicon Panels are used to create targeted NGS libraries for research studies. Whether you need to examine hundreds or thousands of targets, the xGen Custom Amplicon Panels can be tailored to your specific research needs. Once created, the panel is used in a simple, one-tube workflow that is compatible with high-throughput applications.
Table 1. Product specifications of the xGen Custom Amplicon Panels
|Input DNA range||10–25 ng of amplifiable DNA|
|Amplicon size||Versatile size for compatibility with many research sample types: whole blood, cell culture, FFPE, cfDNA (default maximum is 150 bp)|
|Custom design coverage||>90% of requested bases|
|Workflow time||Single-tube with 2-hour DNA-to-library workflow|
|Components provided||Custom primer pool|
|Limit of detection||As low as 1% allele frequency for variants|
|Panel size range||15–1500 amplicons per panel; other options available upon request|
|Library multiplexing capability||Depends on type of indexing primers used|
|On-target %||>90% on-target reads|
|Coverage uniformity||>80% coverage uniformity at a >0.2 of mean depth|
From DNA to NGS library in as little as 2 hours, the workflow for the xGen Custom Amplicon Panels can be done in a single tube with minimal hands-on steps. As shown in Figure 2, the DNA sample is first amplified with the multiplex custom panel to create copies of each of your intended targets. Then the amplicons are converted into indexed libraries in a second PCR reaction with the appropriate indexing primers for your NGS instrument. When multiple libraries are pooled, IDT-proprietary xGen Normalase reagent is used to normalize the libraries for NGS analysis.
Figure 2. xGen Custom Amplicon Panels have a single tube workflow that is done in as little as 2 hours. Creating an NGS library starts with multiplex PCR. Your custom panel is combined with the DNA sample to amplify the targets of interest. The samples are then amplified with indexing primers to create a functional dual indexed library. As an optional step, the xGen Normalase reagent can be used after pooling multiple libraries to ensure each is equally represented in the final sample for the flowcell.
Our xGen Custom Amplicon Panels are compatible with (but not limited to) the following sample types:
Low library yields and primer dimers may indicate one or more of the following:
There are a few key considerations when analyzing sequencing data generated from the xGen HS EGFR Pathway Amplicon Panel with unique molecular identifiers (UMIs).
The first 10 bases in front of Read 2 constitute a UMI. For these first ten bases we recommend trimming them with Trimmomatic (using the CROP option) to make an MID/UMI fastq file for use with the MID pipeline from the fgbio package (Fulcrum Genomics). Before aligning the reads, make sure that the 10 bp UMI (which contains random bases) has been trimmed from 5’ of Read 2.
Also, check that adapter trimming is enabled while setting up the sequencing run. Alternatively, adapter trimming can be performed bioinformatically before analysis.
xGen Custom Amplicon Panels are designed with overlapping amplicons to provide contiguous regions of coverage in a single-tube format. Synthetic primer sequences will be encountered both at the beginning and end of some reads which must be trimmed during analysis. This can be done using a publicly available tool called Primerclip. See our app note Primerclip—A Tool for Trimming Primer Sequences for detailed information.
For more advice, reach out to our Scientific Application Supports team.
Note: A target BED file is provided with purchase of the xGen HS EGFR Pathway Amplicon Panel or the xGen Custom Amplicon Panel.
Please contact Scientific Application Support team if you would like assistance confirming compatibility of your own primers with the xGen Amplicon Sequencing workflow.
Note that using your own UDI primers without Normalase™ modifications will make the amplicon libraries incompatible with the downstream Normalase workflow.
Contact your local sales representative, distributor, or our Scientific Application Support team if you would like assistance in ordering custom Normalase™ indexing primers for the xGen Amplicon workflow.
*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.