Estimating TMB scores and MSI status using the Biomedical Genomics Analysis plugin for CLC Genomics Workbench
In immuno-oncology research, tumor mutation load is a promising marker with the potential to guide immunotherapeutic decisions (1). NGS-based approaches are the preferred methods to interrogate this marker in DNA extracted from tumor biopsies.
The QIAseq Tumor Mutation Burden Panel has been designed to target variants in 486 genes related to tumor and immune biology, covering a total 1.3 Mbp of DNA with Single Primer Extension (SPE) technology. The panel makes use of Unique Molecular Indices (UMIs), supporting sequencing error correction and reduction of PCR amplification bias during bioinformatic analysis. After DNA extraction, library preparation and sequencing, the NGS reads can be analyzed using the QIAseq Tumor Mutational Burden (TMB) ready-to-use bioinformatics workflows delivered with the Biomedical Genomics Analysis plugin for CLC Genomics Workbench.
Figure 1. Coverage of the QIAseq Tumor Mutational Burden Targeted TMB Panel.
Through a series of tools and filters, the TMB workflow reports genetic variants detected, computes the TMB score and provides a genome viewer for convenient investigation of variants and read mappings.
The TMB scores computed by the workflow correlate closely to whole exome sequencing (WES) results reported in two recent quality assurance initiatives focusing on TMB score harmonization across academic centers, pharma companies and diagnostic service providers (2, 3) (Figure 1).
The microsatellite instability (MSI) status of the sample can also be assessed if the MSI booster panel (cat no. SDHS-10101-11981Z-48) is used during sample preparation. The MSI algorithm compares the length profiles of selected microsatellite markers in the sample to reference profiles to assess the stability status at each MSI locus and in aggregate for all loci for the sample. A reference profile comes with the software; however, it is recommended that users construct their own reference profiles using tools provided. This is encouraged, as sequencing chemistries and laboratory procedures used may introduce experimental variation not captured in the provided reference profiles. For MSI calling, the nine quasimonomorphic mononucleotide repeats of the panel give the most consistent results, as also recommended by Buhard et al (4).
The workflows allow for calling copy number variants (CNVs) if appropriate normal reference samples are available and specified in the workflow.
Controlling for quality in the analysis workflow
UMI consensus reads are generated from the data. Using the mapping of these reads against the reference sequence forms the basis of the subsequent analysis, substantially reducing false-positive variant calls. In addition, filtering steps applied at several levels ensure that only quality results of interest are reported. These steps include removing reads originating from homologous genes and pseudogenes from the read mapping, and, after low variant frequency detection, removing variants likely to be artifacts, such as low-quality variants and germline variants (homozygous variants and variants considered common in the population as found in dbSNP). Only variants called at sites of 100X coverage or more are included in the final number, and the TMB score is then calculated by dividing with the sequence length of the panel covered at minimum 100X in mega base pairs (MBP).
The variants reported by the workflow can then be submitted to QIAGEN Clinical Insight (QCI) Interpret to identify pathogenic variants.
Figure 2. Variability in TMB estimates for each tumor cell line across all 15 participating laboratories (4). The QIAseq Tumor Mutation Burden Panel is LAB 12.
Data quality and TMB analysis
It should be noted that TMB can only be reliably scored on high-quality samples. The quality of the sample can be evaluated prior to library construction by checking the amount and purity of DNA obtained after extraction, as well as ensuring that the DNA fragment size peak after fragmentation is 300–350 bp. High-quality samples will have more than 1 MB of target region with greater than 100X coverage. This measure is reported in the QC for Targeted Sequencing report and the TMB report. Another indicator of high-quality samples is the average number of reads per UMI, reported in the UMI Group Report. This value should be between 2 and 4. Lower values (e.g., 1) or higher values (e.g., >5) are indicative of poor samples (too little DNA input, fragments are too short, or both). For other recommendations relating to QC values, please refer to the manual.
Availability and customization
Ready-to-use QIAseq TMB analysis workflows tailored for Illumina and for Ion Torrent are delivered with the Biomedical Genomics Analysis plugin for CLC Genomics Workbench and via a CLC Genomics Server plugin, supporting those with many samples to analyze, such as a hospital or other enterprise setting. By running analyses on CLC Genomics Server nodes, either dedicated job nodes or those of an existing HPC cluster, multiple samples can be analyzed in parallel.
As for all ready-to-use workflows, these workflows can be easily customized, as described in this tutorial. The TMB and MSI calling parts of the workflows are generic, and can be adapted to any other DNA panel workflow. For the development of TMB analysis workflows for Illumina's TSO500 or panels from other vendors, please contact our Custom Solutions team.
References
1. Samstein R. et al. (2019) Tumor mutational load predicts survival after immunotherapy across multiple cancer types. Nature Genetics 51, 202–206.
2. Stenzinger A. et al. (2019) Tumor mutational burden standardization initiatives: Recommendations for consistent tumor mutational burden assessment in clinical samples to guide immunotherapy treatment decisions. Genes Chromosomes Cancer 58, 578–588. doi:10.1002/gcc.22733
3. Friends of Cancer Research [press release]. Friends of Cancer Research Announces Launch of Phase II TMB Harmonization Project. September 18, 2018. https://www.focr.org/news/friends-cancer-research-announces-launch-phase-ii-tmb-harmonization-project
3. Merino DM. et al. (2019) TMB Standardization by Alignment to Reference Standards Phase 2 of the Friends of Cancer Research TMB Harmonization Project. ASCO Meeting Library; Presented Saturday, June 1, 2019.https://meetinglibrary.asco.org/record/172797/abstract
4. Buhard O. et al. (2004) Quasimonomorphic Mononucleotide Repeats for High-Level Microsatellite Instability Analysis. Disease markers 20, 251–257. doi:10.1155/2004/159347.
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At the AGBT conference in Orlando we announced a new collaboration with 10x Genomics to co-market and co-develop NGS, single-cell biology, and bioinformatics solutions. We believe that 10x Genomics will be innovative collaborators in the advancement of human genetics, and our partnership will unfold in multiple phases.
The collaboration will include an investigation of the implementation of 10x Genomic’ GemCode technology with our GeneReader NGS system. And from a bioinformatics perspective, we’ll also look into enabling the processing and analysis of 10x Genomics’ “Linked-Reads” with our suite of bioinformatics solutions aiming to address the challenges of data analysis, interpretation, clinical decision-support, and reporting that rely on testing for long-range phasing and structural variants in genomics.
We are thrilled to collaborate with 10x Genomics, and we look forward to providing even more comprehensive solutions for our NGS customers.
For more information the collaboration, please read the official press release below.
Press release
QIAGEN and 10x Genomics to enter into co-marketing and co-development collaboration
Multi-phase collaboration to advance next-generation sequencing, single-cell biology, and bioinformatics Sample to Insight solutions
Orlando, Florida, February 9, 2016 – QIAGEN N.V. (NASDAQ: QGEN; Frankfurt Prime Standard: QIA) today announced a collaboration with 10x Genomics to develop and promote comprehensive solutions for next-generation sequencing (NGS), single-cell biology and bioinformatics.
This collaboration includes:
“QIAGEN’s collaboration with 10x Genomics enables both companies to better serve its customers, helping them make groundbreaking advances in single-cell biology, bioinformatics and beyond,” said Peer Schatz, CEO of QIAGEN. “10x Genomics is recognized for delivering critical long-range sequence information that greatly expands the quality of genome research. This partnership will further the potential of each of our product lines while providing significant new capabilities to NGS researchers.”
“We welcome the opportunity to partner with QIAGEN, with their comprehensive offerings for genomic analysis, ranging from sample preparation technologies to bioinformatics solutions for the interpretation and analysis of genomic data,” said Serge Saxonov, 10x’s Chief Executive Officer. “Access to long-range sequence information is becoming the standard for obtaining the most comprehensive understanding of disease, and we expect this collaboration to allow our respective customers to gain valuable insights that have previously not been possible.”
10x Genomics’ GemCode™ Platform overcomes existing NGS limitations through a combination of microfluidics, chemistry and bioinformatics. The system is a unique molecular barcoding and analysis platform that consists of instrumentation, reagents and software to provide a complete approach for a broad range of applications, including single-cell transcriptomics and whole genome analysis.
About QIAGEN
QIAGEN N.V., a Netherlands-based holding company, is the leading global provider of Sample to Insight solutions to transform biological materials into valuable molecular insights. QIAGEN sample technologies isolate and process DNA, RNA and proteins from blood, tissue and other materials. Assay technologies make these biomolecules visible and ready for analysis. Bioinformatics software and knowledge bases interpret data to report relevant, actionable insights. Automation solutions tie these together in seamless and cost-effective molecular testing workflows. QIAGEN provides these workflows to more than 500,000 customers around the world in Molecular Diagnostics (human healthcare), Applied Testing (forensics, veterinary testing and food safety), Pharma (pharmaceutical and biotechnology companies) and Academia (life sciences research). As of December 31, 2015, QIAGEN employed approximately 4,600 people in over 35 locations worldwide. Further information can be found at http://www.qiagen.com.
Certain of the statements contained in this news release may be considered forward-looking statements within the meaning of Section 27A of the U.S. Securities Act of 1933, as amended, and Section 21E of the U.S. Securities Exchange Act of 1934, as amended. To the extent that any of the statements contained herein relating to QIAGEN's products, markets, strategy or operating results, including without limitation its expected operating results, are forward-looking, such statements are based on current expectations and assumptions that involve a number of uncertainties and risks. Such uncertainties and risks include, but are not limited to, risks associated with management of growth and international operations (including the effects of currency fluctuations, regulatory processes and dependence on logistics), variability of operating results and allocations between customer classes, the commercial development of markets for our products in applied testing, personalized healthcare, clinical research, proteomics, women's health/HPV testing and nucleic acid-based molecular diagnostics; changing relationships with customers, suppliers and strategic partners; competition; rapid or unexpected changes in technologies; fluctuations in demand for QIAGEN's products (including fluctuations due to general economic conditions, the level and timing of customers' funding, budgets and other factors); our ability to obtain regulatory approval of our products; difficulties in successfully adapting QIAGEN's products to integrated solutions and producing such products; the ability of QIAGEN to identify and develop new products and to differentiate and protect our products from competitors' products; market acceptance of QIAGEN's new products, the consummation of acquisitions, and the integration of acquired technologies and businesses. For further information, please refer to the discussions in reports that QIAGEN has filed with, or furnished to, the U.S. Securities and Exchange Commission (SEC).
