NGS Reference Standard – MEDGENOME

Archana Deshpande, QA Manager, MedGenome Inc


Next-generation sequencing (NGS) data is increasingly used in clinical diagnostics to identify genetic variants that may cause disease. A major challenge in using NGS data in the clinical setting is correct interpretation due to their enormous size and complexity. Also, technical errors can occur during the sample processing and/or sequencing steps inherent in the type of sequencing technique used. The use of reference standards is therefore paramount to mitigating and minimizing these errors.

Reference standards play an important role in the life cycle of a typical NGS method prior to clinical application. A typical NGS assay life cycle includes assay development, optimization, validation, and ongoing quality control. Standards are important in all these aspects, from assay validation to technical validation to sample processing. In this article, we discuss the general selection of these reference standards and detail the results of the technical validation of target capture assays (His TruSight Oncology 500 panel from Illumina) performed at MedGenome Labs.

NIST and GIAB standards

There are several consortia like GIAB (Genome in a Bottle) and companies (Horizon Diagnostics and SeraCare) that have developed DNA reference materials over the years to support clinical translation of whole genome sequencing. . NIST (National Institute of Standards and Technology) also had a program to develop a Whole Human Genome Reference Material. Reference standards are generally well-characterized samples that are consistent and stable over time. In essence, DNA referencing is characterized by collating data from a variety of sequencing and bioinformatics methods and multiple datasets to produce highly reliable genotype calls. This data can be used by laboratories to evaluate assay performance and by accredited laboratories to evaluate benchmarking results.

Besides being homogeneous and stable, NIST defines standards with certified values ​​that indicate confidence in their accuracy. This accreditation indicates that NIST has thoroughly investigated and described all known or suspected sources of bias in the data.

MedGenome validation data

MedGenome Labs validated Illumina’s TSO 500 workflow and pipeline using SeraCare reference samples. The TSO 500 is a targeted capture-based panel investigating multiple biomarkers and tumor types. Identify all relevant DNA and RNA variants associated with various types of solid tumors. Thus, in-house comprehensive genomic profiling of tumor samples is possible. It also accurately measures microsatellite instability (MSI) and tumor mutational burden (TMB), the current leading cancer immune biomarkers. Another advantage is that the assay has a liquid biopsy-ready ctDNA panel.

Both TSO 500 workflows are hybrid capture protocols and three control ctDNA from SeraCare (different allele frequencies 0.1%, 0.5% and wild type) were used to validate the process. The data generated were from an initial input of only 30 ng.

Analysis of somatic mutations was performed using the gene list present in Seraseq ctDNA Complete Mutation Mix AF 0.5%, AF 0.1% and WT. Analyzes included sensitivity, specificity, positive predictive value, and run-to-run comparisons. Below are some of the results obtained.

library quality report

Libraries generated from SeraSeq controls ranged from 23 to 46 nM with an average size of 330 bp and an insert size of ~200 bp (see Figure 1 for an example). The size of this library is within the range specified by the TSO 500 protocol.

Figure 1: Example of final library from SeraSeq ctDNA control (which had an allele frequency of 0.5% with a peak at 334 bp)

The reference SeraSeq set was analyzed using the TruSight Oncology 500 ctDNA Local App. Results were compared with data from a vendor-supplied reference set. We were able to obtain 100% sensitivity and specificity for all three controls in the dataset. Variants are also represented in lollipop plots shown below along with sensitivity and specificity information for controls (Table 1).

Lollipop plot showing gene variants

For run-to-run comparisons, samples were compared for tumor mutation burden and variant allele frequency (VAF) for the same sample between two runs. The identified variants were found to be nearly identical and the VAF values ​​are highly correlated (see Figure 2 below).

Figure 2: VAF comparison chart for run-to-run comparison

Illumina’s TSO 500 panel process workflow has passed technical validation and we are now offering TSO500 panels for both solid tumors and ctDNA as part of our service to our clients..


DNA reference standards are essential for research as well as translational medicine. MedGenome uses commercially available reference standards and performs technical validation whenever available. Validation gives you confidence in your process workflow and generated data.


    1. 1. Genomic reference materials for clinical application Justin Zook and Marc Salit Biosystems and Biomaterials Division, National Institute of Standards and Technology, 100 Bureau Dr., Gaithersburg, MD 20899
    2. 2. Reference standard for next-generation sequencing Simon A. Hardwick, Ira W. Deveson, Tim R. Mercer, Nature Reviews Genetics June 2017

#NGS data, #NGS, #benchmarks, #standards, #TruSight oncology, #TSO500, #immune-oncology biomarkers, #microsatellite instability, #tumor mutation burden NGS Reference Standard – MEDGENOME

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