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You can purchase standalone library prep reagents or a kit that includes both the library prep and sequencing reagents. The following kits are available:
You can order an additional MiSeq reagent kit v3 (600 cycle) (MS-102-3003) or MiniSeq High Output reagents (300 cycle) (FC-420-1003).
TruSight Tumor 15 targets 15 solid tumor genes, while TruSight Tumor 26 targets 26 genes. TruSight Tumor 15 is a multiplex, PCR amplicon-based assay and the TruSight Tumor 26 uses the TruSeq Custom Amplicon extension-ligation chemistry.
Visit the Safety Data Sheets (SDS) page and enter the applicable search terms.
Expiration dates are printed on the kit box labels and reagent tubes. Illumina guarantees three months of shelf life from the time of kit shipment.
The Illumina Adapter Sequences Document lists index adapter sequences and all other adapter sequences.
The following table lists the genes and exons included in the panel.
Gene | Target or Region | Potential Disease States |
AKT1 | Exon 3 (partial) | Breast |
BRAF | Exon 15 (partial) | Melanoma, Colon, Lung |
EGFR | Focal
Amplification, Exon 12 (partial), 18, 19, 20, 21 (partial) | Lung |
ERBB2 | Focal Amplification, Exons 14
(partial), | Breast, Lung |
FOXL2 | Exon 1 (partial) | Ovary |
GNA11 | Exon 5 (partial) | Melanoma |
GNAQ | Exon 5 (partial) | Melanoma |
KIT | Exons 8, 9, 10, 11, 13, 14, 17, 18 | Gastric, Melanoma |
KRAS | Exon 2 (partial), 3 (partial), 4 | Colon, Gastric, Lung |
MET | Focal Amplification | Lung, Colon, Gastric |
NRAS | Exon 2 (partial), 3 (partial), 4 | Colon |
PDGFRA | Exon 12, 14, 18 | Gastric, Melanoma |
PIK3CA | Exon 10, 21 | Lung, Breast, Prostate |
RET | Exon 16 | Lung |
TP53 | Full CDS | Lung, Melanoma, Ovary, Colon |
Yes, this kit contains dual index adapters - 24 i7 indexes and 2 i5 indexes.
The i7 index is 6 bp and i5 index is 8 bp. Although numbering of indexes seems similar to other kits, the index sequence is different and can be found in the reference guide.
The output predefined report includes the following genes. These genes are also listed on the TruSight Tumor 15 product page.
Gene | Target or Region | Potential Disease States |
AKT1 | E17K | Breast |
BRAF | V600E/K/R/M/D/G | Melanoma, Colon, Lung |
EGFR | Exon 19 and
Exon 20-insertions, deletions, and indels G719A/C/S, L858R, L861Q, S7681, T790M | Lung |
ERBB2 | p.E770_A771insAYVM | Breast, Lung |
FOXL2 | C134W | Ovary |
GNA11 | Q209L | Melanoma |
GNAQ | Q209L | Melanoma |
KIT | Exons 9, 11, 13, 14, 17 | Gastric, Melanoma |
KRAS | Codons 12, 13, 19, 59, 61, 117, 146 | Colon, Gastric, Lung |
MET | N/A | Lung, Colon, Gastric |
NRAS | Codons 12, 13, 59, 61, 117, 146 |
Colon |
PDGFRA | Exons 12, 14, 18 | Gastric, Melanoma |
PIK3CA | Exons 9, 20 | Lung, Breast, Prostate |
RET | M918T | Lung |
TP53 | Full CDS | Lung, Melanoma, Ovary, Colon |
The following FFPE DNA extraction methods have been verified.
These methods are provided in the reference guide.
Both pools target different regions of the genome. Yield and read depth can vary between the two pools, especially if there is an amplification event or large indel. To minimize differences in the 2 pools, all libraries are normalized to 5 ng/ul before sequencing. There could be a slight bias if an amplification event occurs.
The gene specific primers range between 18–35 bps, and most are between 20–25 bps. The tags are 20 and 21 bps long.
We recommend input of 20 ng per sample (10 ng for amplicon mix A and B for each sample) and minimum input DNA concentration of 2ng/µl per amplicon mix.
A fluorometric-based method such as AccuClear (preferred), Qubit, or PicoGreen is recommended for quantification of both input DNA and final libraries.
Yes. This kit has been optimized for FFPE DNA samples.
Dilute starting material in RNase/DNase-free water or 10 mM Tris-HCl, pH 8.5. Avoid EDTA containing buffers, such as TE or AE.
DEPC water is not recommended. Contaminants can inhibit parts of the reaction.
Expect the library concentration of library mix A and B for each sample to be ≥ 20 ng/µl. Libraries with lower yield give poor quality, less accurate variant calls. Some FFPE samples do not yield sufficient library quantity for sequencing.
The typical yield observed is 20–100 ng/µl. The official specification is at least 20 ng/µl.
Process at least four samples per library prep batch.
The following thermal cyclers are recommended for the TruSight Tumor 15 kit. Follow the recommended ramp rate setting listed in the TruSight Tumor 15 Reference Guide
Bio-Rad C1000, Applied Biosystems GeneAmp PCR System 9700, and Eppendorf MasterCycler ep Gradient were qualified internally.
A generic 96-well PCR plate is listed in the reference guide. See the plate recommendation for a specific thermal cycler.
Libraries can be stored for up to 14 days at -25°C to -15°C.
The amplicons are on average ~150-175 bp and the libraries sizes are around 350 bp, but represent a pool of multiplexed PCR amplicons, not a single product. Additionally, primer dimer may be visible in the gel as a double band at around 160-180 bp.
Quantify your libraries using qPCR according to the Illumina Sequencing Library qPCR Quantification Guide.
Quantification with a fluorometric method specific to double stranded DNA is also sufficient.
TruSight Tumor 15 is only supported on the MiniSeq, MiSeq (v3 chemistry), and MiSeqDx (v3 chemistry) Systems. Sequencing on other systems is possible, but has not been tested.
Set up a 2 x 151 bp run.
MiSeq with v3 reagents: Pooling 8 DNA samples (16 libraries) is recommended. However, there are sufficient indexes provided for preparing 24 DNA samples (48 libraries).
MiniSeq: With High Output reagents, pooling 8 DNA samples (16 libraries) is recommended. With Mid Output reagents, pooling 3 DNA samples (6 libraries) is recommended.
The MiSeq v3 and MiniSeq reagent cartridges each contain all required sequencing primers.
The following loading concentrations have provided optimal cluster density. Adjust the loading amount based on the needs of your lab.
We recommend spiking in ~1% PhiX, corresponding to 20 µl of a 20pM denatured PhiX stock in 980 µl DAL.
Two manifest files (one per pool) are required to analyze sequencing runs. The manifest files are installed automatically on MiSeq (MCS/MSR 2.6 or later) or Local Run Manager.
Manifest files can be downloaded from the support pages Downloads section.
VariantStudio can be used for annotation and filtering.
Currently, there are no recommended third-party software tools.
Follow the instructions in the Local Run Manager Software Guide.
These regions are used for normalization of amplification analysis for EGFR, ERBB2, and MET. This amplification software will be released at a later date.
Included variants are available in the report definition file. Download the file from TruSight Tumor 15 Product Files.
The genome.vcf file is the main output file for variant calling. For further variant filtering, use BaseSpace Variant Interpreter or the predefined PDF report.
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