TruSeq Custom Amplicon FAQs

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  • Assay Design


  • Yes. The Avoid SNPs design option also avoids indels from the given variant database being used when possible (eg dbSNP for human designs).

    For important regions with known indels, Illumina recommends inspecting the probes using the linked UCSC tracks per item in the grid. In certain cases, probes overlapping indels can be the best or only option to provide coverage of that region.

    The maximum number of rows allowed is 1000. You can submit multiple files per project.

    A gap equivalent to a maximum amplicon size is required between target regions. Regions with a smaller gap size are merged to improve design performance and prevent unfavorable probe to probe interactions.

    The following table shows the maximum and minimum amplicon lengths for a given amplicon size setting.

    Setting

    Minimum

    Maximum

    150

    125

    190

    175

    170

    190

    250

    225

    275

    425

    400

    450

    Homologs: Having homologs in the same design can lead to low designability. Split homologs into separate CAT pools.

    GC Content: Regions with greater than 80% GC content can be difficult to design against, particularly when these regions are greater than 500 bp in length.

    Homopolymer Sequences and Repetitive Elements: DesignStudio avoids these regions to make sure that probes have better specificity in the genome.

    Poor Specificity: DesignStudio will assess the specificity of probes and exclude those which will not provide satisfactory on-target coverage.

    When Avoid SNPs is turned on, DesignStudio considers the location of polymorphisms and avoids them when possible. If this is not possible, it will place the probes in regions that do not interfere with their binding.

    A primary determinant is the total size of the target regions of interest. In general, smaller custom designs (up to ~0.5 Mb) are optimal for TruSeq Custom Amplicon based approaches and are best suited for sequencing on the MiSeq or NextSeq system.

    Nextera Rapid Capture Custom Enrichment is a better match for mid- to large-size custom designs (~0.5–15 Mb) and pairs best with a HiSeq, NextSeq, or MiSeq System.

    Optimal probes are chosen using an algorithm that considers melting temperature (Tm), % GC, length, secondary structure, uniqueness in the genome, and the presence of underlying SNPs (based on dbSNP). For more information, see the DesignStudio online help.

    Variant calling is only performed in the amplicon region lying between the upstream and downstream probe locations. The probe regions are not included in variant calling. MiSeq Reporter uses the manifest file for each design to avoid calling variants in probe regions that are synthetic oligonucleotides and not biologically relevant.

    • Increase the size of the target. Increasing the size of the target to design against can rescue regions that appeared to be undesignable. The increased size of a target gives DesignStudio a little more flexibility to fit a higher scoring amplicon over the desired target bases.
    • Use the Avoid SNP feature. With this option, DesignStudio is more aggressive when placing amplicons by putting less importance of the location of SNPs in the design process. This can help rescue undesignable regions if DesignStudio avoids regions with low frequency or poorly annotated SNPs in the database, leading to low designability. DesignStudio uses dbSNP 138 or 1000 Genomes (human hg19), SNP 128 (mouse mm9), SNP 125 (rat rn4), and Ensembl UMD3.1 SNPs (bovine) to position probes that avoid SNP and indel locations to improve accuracy and performance of the design for that region.
    • Change the context of the panel. For example, putting a highly homologous or high GC rich target sequence into the same multiplex design can be problematic for designing probes to amplify each target discretely. Moving problematic regions into a separate design can frequently improve the designability.
  • Input


  • Commercial or lab-validated DNA extraction methods typically yield DNA that is compatible with this assay. DNA purity must have an A260/A280 ratio of 1.8–2.0. PicoGreen is recommended.

    If you are using FFPE DNA at the supported amplicon sizes (150 or 175 bp amplicons), follow the recommended QC steps to select DNA of appropriate quality.

    DesignStudio accepts the following inputs:

    • Chromosome number with location of starts and stops
    • Gene name (with an option for exons only and upstream and downstream bases)
    • Batch upload with a *.csv file. 

    Templates for coordinates and genes are provided in the file upload section for adding new target regions. For more information, see the DesignStudio Online Help.

    Only use FFPE-derived DNA when using short amplicon lengths of 150 bp or 175 bp. Shorter amplicons provide better amplification than longer ones when the sample input is fragmented FFPE-derived DNA. To run library prep with FFPE samples, use the Illumina TruSeq FFPE DNA Library Prep QC Kit to determine input amount based on ∆Cq.

    This kit enables targeting of ~650 kb of cumulative DNA sequence (1536 amplicons × 425 bp each = ~650 kb).

  • Protocol


  • The assay takes less than 8 hours total, with 2–3 hours of hands-on time. The libraries are then immediately compatible with the MiSeq System without any further manipulation.

    CAT is a custom amplicon oligo tube, which is the final pool of oligonucleotide probes used to generate amplicons in the assay. One CAT supports the amplification of up to 1,536 amplicons in a reaction.

    An amplicon is a fragment of DNA that is produced by an amplification event such as PCR.

    Amplicon sizes can be 150, 175, 250, or 425 bp. Sizes are user-defined when creating a project in DesignStudio.

    Yes. The number of PCR cycles has been optimized for various assay conditions, taking into account both the number of amplicons per reaction and the type and amount of input DNA (eg, FFPE). The recommended number of PCR cycles ensures sufficient yield for the bead-based normalization while avoiding overamplification.

    Ideally, perform pre-PCR and post-PCR procedures in separate rooms. If separate equipment is used (eg, shaker, heat block, centrifuge) and a strict cleaning schedule is maintained, then the assay can be performed in designated, nonadjacent areas in the same room. Regular bleaching is outlined in the reference guide. In addition, filtered pipette tips are also recommended.

    Currently, this kit supports up to 1536 amplicons in a single reaction. The number of amplicons in the multiplex reaction varies based on the custom design and can range from 16–1536 amplicons. If more amplicons are desired (eg, 3072), two reactions can be used with the same genomic DNA sample input. A single user can easily prepare more than ~150,000 sequencer-ready amplicons in a single day (96 samples × 1,536 amplicons).

    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. Using indexed primers supplied with the kit, DNA templates are then PCR amplified, pooled into a single tube, and sequenced on the MiSeq System. For additional details on the assay, see the assay reference guide.

  • Analysis


  • The following software tools are available for this kit: Illumina Experiment Manager, MiSeq Reporter, and the Illumina Amplicon Viewer.

    MiSeq Reporter is a simple, on-instrument tool that demultiplexes samples, performs alignment of reads, and creates a variant report immediately upon completion of each sequencing run. MiSeq Reporter also creates an assembly for each amplicon (on a per-sample basis), performs per-SNP calling, and generates graphs and reports, including the sequencing coverage per amplicon.

    Illumina Amplicon Viewer performs data visualization from multiple runs and custom report generation off the instrument. Analysis workflows are available for detecting both germline and somatic variation in sequenced samples.

    DesignStudio returns high-confidence amplicon designs that have delivered unprecedented amplicon multiplexing performance. You can expect to see specificity > 70% and uniformity > 80%. In practice, we have observed specificity and uniformity > 90% for hundreds of designs.

    No. File directory structures from a HiSeq system are incompatible with MiSeq Reporter software.

    However, the TruSeq Amplicon App is available in BaseSpace and can be used to analyze the this kit.