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Visit the Safety Data Sheets (SDS) page and enter the applicable search terms.
The TruSight One *.bed file details all regions of interest (ROI) and lists all targets of enrichment for this assay. Filter by gene name (HGNC nomenclature), or reference the coordinates of your loci of interest in the human genome reference (hg19 build). See the product files for the gene list.
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.
Illumina recommends using a fluorometric-based method that is specific to double-stranded DNA, such as QuantiFluor or PicoGreen. For more information, see the input recommendations in the TruSight One Library Prep Guide.
Overloading the Nextera tagmentation reaction with more than 50 ng of genomic DNA leads to a larger library size distribution, which can lower the number of on-target sequencing reads. Less than 50 ng of input DNA leads to a smaller library size distribution. The result is reduced library diversity or elevated duplicate reads because many of these smaller library sized fragments would be eliminated during the size-selection procedures.
You can download the TruSight One manifest file from TruSight One Sequencing Panel Kit Product Files.
When quantifying the post-enriched library using a fluorometric method, clustering at 12.5 pM generates cluster densities ranging from 1200–1400 K clusters/mm2 using the MiSeq v3 software and reagents.
Results vary based on quantification method and instrument-to-instrument variability. For guidance, see the Cluster Optimization Overview Guide.
You can sequence TruSight One libraries on any Illumina sequencing system.
A trio of TruSight One-enriched sample libraries can be sequenced so that >95% of the target regions are covered at 20x minimum coverage. This coverage can be achieved using the 300-cycle MiSeq v3 reagent cartridge included with the 9-sample TruSight One kit.
To allow the flexibility to prepare 12-plex library pools that target depths with one rapid flow cell sequenced on the HiSeq 2500 System.
The TruSight One assay is designed for sequencing with a 2 x 151 bp read length to maximize coverage of the targeted region. The design minimizes the likelihood of sequencing into the uninformative adapter sequence.
TruSight One libraries use dual indexes and additional 8-cycle Index 1 and Index 2 reads to demultiplex pooled enrichments. For more information on indexing and pooling, see the TruSight One Sequencing Panel Reference Guide.
Three (3) MiSeq v3 reagent cartridges are included with the 9-sample TruSight One Sequencing Panel kit (FC-141-1006). If you need additional MiSeq reagent cartridges, order additional TruSight One 9-sample kits or MiSeq v3 600-cycle kits (MS-102-3003).
Use the Enrichment workflow to create a sample sheet formatted for analysis of TruSight One data. For instructions, see the Illumina Experiment Manager User Guide.
TruSight One regions, targets, and probes are provided in *.bed file format and can be found by visiting the TruSight One Product Support page. The *.bed file can be imported into either the UCSC Genome Browser (genome.ucsc.edu) or the Integrated Genome Viewer (IGV) (www.broadinstitute.org/igv/).
Yes, BaseSpace Sequence Hub can be used for the analysis of TruSight One runs. Illumina recommends using the Illumina Experiment Manager, and selecting Targeted Sequencing and the subsequent Enrichment workflow. Download VCF files from BaseSpace Sequence Hub projects to interpret variants in BaseSpace Variant Interpreter.
Subpanels can be created by generating gene lists in HGNC nomenclature for the regions of interest. The complete TruSight One gene list is available in the product Downloads. To filter variants based on the shortened gene list, upload this subpanel gene list when importing *.vcf files into VariantStudio.
The expected final percentage of data from enriched regions is 50–70%, but varies across enrichments and can be influenced by the quality of starting material.
The enrichment wash steps are key to reducing nonspecific DNA binding and require samples to be maintained at the indicated temperature. Incorrect temperatures can result in lower percent enrichments and decreased yields. Use the proper equipment and make sure that the streptavidin beads are properly resuspended.
User-supplied equipment, temperatures settings, and enrichment wash procedures are provided in the TruSight One Sequnecing Panel Reference Guide.
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