For creating a comprehensive profile of Tumor Mutational Burden (TMB) and Microsatellite Instability Status (MSI)
Create highly uniform libraries
Easy workflow using enzymatic fragmentation
Robust analysis modules
Researchers face the challenge of creating a reliable, consistent workflow for easily processing samples to understand the mutational landscape of tumors. Tumor Mutational Burden (TMB) is the measure of the number of mutations found within a tumor. However, a lack of standardized testing has prevented any meaningful movement to create a TMB biomarker. The new QIAseq Tumor Mutational Burden Panels overcome the challenges of earlier assay designs to create a comprehensive profile of TMB and Microsatellite Instability (MSI) status by achieving high analytical sensitivity, with lower false and negative rates, while still maintaining >95% correlation with whole exome datasets. QIAseq NGS assays, including the QIAseq TMB and MSI Panels, have a fail rate of less than 5% for samples that have passed QC and provide a TMB score. The QIAseq TMB Panel has been tested in several key opinion leader labs and has performed as well or better than many earlier products. This comprehensive panel covers 486 genes and can be boosted to add 27 MSI markers. Want to try this solution for the first time? Request a quote for a trial.
Specifically, the QIAseq TMB Panel:
Correlates well with Whole Exome Sequencing – in silico comparison shows over 98% correlation
Correlates well with established TMB panels – in vitro comparison shows over 93% correlation
Kit containing ALL reagents (except indexes) for targeted DNA sequencing; fixed panel for 12 samples; more than 100 genes
QIAseq Tumor Mutational Burden Panels are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
Isolated DNA, as low as 20 ng, is enzymatically fragmented to generate small pieces of dsDNA. This is followed by the library construction step, in which IL-N7 adapters, molecular barcodes, and sample indexes are incorporated into DNA fragments generated in the previous step. Library fragments now serve as templates for target enrichment using single primer extension. In this step, targets are enriched using a single gene-specific primer and a universal forward primer. The final step is library amplification and sample indexing (for dual indexing) using the IL-S5 sample index primer and a universal primer.
Principle of molecular bar codes
A variant identified in a sample represents one of two events: a true or false variant. False variants can be introduced at any step during the workflow, including sequencing reactions. This results in the inability to accurately and confidently call rare variants (those present at 1% of the sample). Due to PCR duplicates generated in amplification steps, all DNA fragments look exactly the same, and there is no way to tell whether a specific DNA fragment is a unique DNA molecule or a duplicate of a DNA molecule. With molecular bar codes, since each unique DNA molecule is bar coded before any amplification takes place, unique DNA molecules are identified by their unique bar codes, and PCR duplicates carrying the same bar code are removed, thereby increasing the analytical sensitivity of the panel.
The QIAseq Targeted DNA Panels deliver outstanding sequencing metrics. 6000 SNPs were enriched from 20 ng of NA12878 DNA. Library was constructed for sequencing on a MiSeq, with 4,000,000 reads generated. The panel achieved a uniformity of 99.5% at 0.2x of mean coverage, and 96% at 0.5x of mean coverage.
Coverage of GC-rich genomic regions
The QIAseq DNA panels use a proprietary buffer mixture to efficiently sequence GC-rich regions within the genome. Two examples are shown here: CEBPA and CCND1. Complete coverage of exonic regions within those two genes is achieved.
Gene content of QIAseq Tumor Mutational Burden Panels
Profile 486 genes for checkpoint pathways and key oncogenes involved in high mutational load in cancer. Content can be boosted with your custom content and with a comprehensive MSI panel targeting 27 loci.
Accuracy: Innovative digital sequencing (incorporating UMIs) eliminates PCR duplication and amplification artifacts to detect low-frequency variants with high confidence (see Figure "Principle of molecular bar codes").
Analytical specificity: The unique combination of our proprietary primer design algorithm and rigorous testing of every primer assay guarantees high analytical specificity and accurate results.
Uniformity: The QIAseq TMB Panel workflow has been optimized to deliver highly uniform sequencing results, to ensure sequencing capacity is utilized very efficiently (see Figure "Uniformity").
Analytical sensitivity: Digital DNA sequencing approach is optimized to deliver high confidence in calling low-frequency DNA variants. Over 90% analytical sensitivity for 1% NA12878 SNP and indel on typical coding region with false positive less than 15 per mega base region when variants are detected with tiled primer design to cover complete coding region of each gene.
Universality: The chemistry used in the QIAseq TMB Panels and workflow is compatible with both regular and GC-rich genomic regions, allowing one to achieve 100% coverage of genes rich in GC content such as CEBPA and CCND1 (see Figure "Coverage of GC-rich genomic regions").
Flexibility: The QIAseq TMB Panels offer a high degree of flexibility in content and sample multiplexing. Several cataloged panels have been developed for a wide range of applications. One can also build a custom panel for a specific content, or extend the contents of an existing cataloged panel. Up to 384 samples can be multiplexed using the QIAseq indexes.
Performance table QIAseq TMB Panels and MSI:
Number of genes
Analytical specificity (on-target)
Uniformity (0.2x mean depth)
Variant allele frequency for TMB
Types of variants called
TMB score, SNVs, Indels, CNVs
Recommended DNA input
Total workflow time
PCR duplicates are a major issue in targeted DNA sequencing, since, through PCR amplification, they turn unique DNA molecules into identical DNA molecules that cannot be distinguished from each other. In addition, errors from PCR amplification and sequencing process may also be present in final reads that lead to false positive variants in sequencing results. This, in turn, results in the inability to confidently call DNA variants present at low frequencies in the starting DNA material. To overcome the issue of PCR duplicates and amplification artifacts, the QIAseq TMB Panels use digital sequencing by incorporating UMIs into the starting DNA material before any amplification takes place, thereby preserving the uniqueness of the starting DNA molecules and overcoming the issues of PCR duplicates, false positives and library bias.
The entire workflow of the QIAseq TMB Panels to go from extracted DNA to sequencing-ready libraries can be completed in 9 hours (see Figure "Workflow"). Extracted DNA is fragmented, genomic targets are molecularly bar coded and enriched, and libraries are constructed. Sequencing files can be fed into the QIAseq pipeline, a cloud-based data analysis pipeline, which will filter, map and align reads, as well as count unique molecular bar codes associated with targeted genomic regions, and call variants with a bar code-aware algorithm. This data can then be fed into IVA or QCI for interpretation.
The QIAseq TMB Panels can be used to call a variety of DNA variants from a wide range of sample types for numerous applications.
Fresh or frozen tissue
Call TMB scores
Profiling of DNA variants in solid and hematologic malignancies