TruSight Myeloid amplicons are 225–275 bp in length.
The TruSight Myeloid Sequencing Panel targets 54 genes (either full coding sequence or targeted exons) that are frequently mutated in myeloid malignancies. Refer to the TruSight Myeloid Sequencing Panel Data Sheet for a full list of genes.
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.
No. FFPE DNA is not supported.
The assay uses 50 ng intact, high-quality genomic DNA. DNA extracted from Formalin-Fixed, Paraffin-Embedded (FFPE) tissue is not supported.
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.
Yes. The number of PCR cycles has been optimized for the number of amplicons per reaction. The recommended number of PCR cycles for different amplicon numbers is indicated in the user guide and ensures sufficient yield for the bead-based normalization while avoiding over-amplification.
The ACD1 control is included to allow customers to assess the success of the library preparation. Illumina recommends that customers who are new to the assay include the ACD1 control. Technical Support will not be able to troubleshoot without this control. If the ACD1 control is included in the sequencing run, it should replace a test sample. For example, if the assay recommends eight samples per run and the ACD1 control is included, then there will be seven test samples plus the ACD1 control.
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.
A 2 × 150 bp paired-end read is recommended for TruSight DNA amplicon libraries, such as TruSight Myeloid.
TruSight DNA Amplicon libraries are fixed content panels that leverage Illumina’s TruSeq Custom Amplicon (TSCA) assay. TSCA is the fastest and easiest multiplexed amplicon assay optimized for the MiSeq System. It allows for the sequencing of 16–1536 custom amplicons (small regions of interest) across up to 96 samples. The assay also includes on-instrument variant calling for somatic variation with the Somatic Variant Caller on the MiSeq System.
TruSight Myeloid has integrated sample barcodes that enable pooling of up to 96 samples per MiSeq run. However, the actual number of samples that should be pooled together per sequencing run depends on the number of amplicons and the desired depth of sequencing coverage. For TruSight Myeloid libraries, eight samples per MiSeq run with MiSeq v3 reagents should give the appropriate amplicon sequencing depth.
An amplicon is a fragment of DNA produced by an amplification event such as PCR. For TruSight Myeloid libraries, amplicon sizes are fixed at a length of 225–275 bp.
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 TruSight DNA Amplicon can be performed in designated, non-adjacent areas in the same room. Regular bleaching is outlined in the User Guide. In addition, filtered pipette tips are also recommended.
Yes, but you will see lower coverage depth per sample as a result.
You do not need an upgraded MiSeq System to run these samples. However, consider the coverage needed for your study when designing an experiment. An online calculator is provided in DesignStudio to help with these calculations.
A TSO is a TruSight oligo, which is the final pool of oligonucleotide probes used to generate amplicons in the assay. One TSO supports amplification of all amplicons in a reaction.
Illumina recommends the following to give > 95% of amplicons at > 500x coverage:
For an example of data from a TruSight DNA Amplicon experiment, refer to the data set available via BaseSpace. This sample data set is from the TruSight Myeloid Sequencing Panel sequenced on the MiSeq with MiSeq v3 reagents.