DesignStudio accepts the following inputs:
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.
We recommend 10 ng high quality genome DNA. For FFPE samples the input amount depends on ∆Cq. For more information, see the TruSeq FFPE DNA Library Prep QC Reference Guide (document # 1000000002137).
TruSeq Custom Amplicon enables targeting of ~384 kb of cumulative DNA sequence (1536 amplicons × 250 bp each = ~384 kb).
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.
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.
Currently, TruSeq Custom Amplicon Low Input 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. A single user can easily prepare more than ~150,000 sequencer-ready amplicons in a single day (96 samples × 1,536 amplicons).
See TruSeq Custom Amplicon Low Input Library Prep Reference Guide for more details about the new workflow.
An amplicon is a fragment of DNA produced by an amplification event such as PCR.
Amplicon sizes can be 150, 175, or 250 bp. Sizes are user-selected when creating a project in DesignStudio.
Use the Qiagen QIAamp DNA FFPE Tissue Kit (catalog # 56404).
A 2 × 150 bp paired-end read is recommended on the MiSeq System for 150–250 bp amplicon sizes.
Yes, they are different. TruSeq Custom Amplicon Low Input manifest files are available on the Product Files page.
Analysis can be performed using the TruSeq Amplicon BaseSpace App or MiSeq Reporter TruSeq Amplicon Workflow.
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.