Confirmation of NGS for False-negative Variants Using XNA Technology

Is it necessary to confirm variants from NGS?

Next-generation sequencing (NGS) is a powerful tool that has seen a fast increase in clinical labs although only a few NGS tests have been approved by the FDA. However, there have been a lot of debate on if variants from NGS sequencing should be confirmed either by Sanger sequencing, the gold standard, or other techniques such as quantitative PCR, or the combination, or other methods. Although we see a guideline for somatic variant detection using NGS is available in January 2018 from the Department of Health in New York State mentions about confirmation, guidance on laboratory standard in term of NGS confirmation on NGS is different from different organizations, such as College of American Pathology (CAP) and American College of Medical Genetics and Genomics (ACMGG).

A report in 2016 with 8000 Sanger-sequenced variants from 20,000 clinical samples found 1.3% false-positive variants. The author strongly suggested confirming NGS false-positive mutations although there it is required for clinical labs. Although confirmation of NGS mutations will add cost and delay for results, it is important to have accurate gene sequence information for patients’ next step therapy strategies.

False-positive mutations are not preventable in NGS

False-positive mutations are generated in NGS due to the sample preparation steps such as enrichment, instrument use, and bioinformatics analysis steps. They often occur in the AT-rich, GC-rich, or homopolymer stretches. Although the false-positive variants can be set to zero by adjustment of the bioinformatics pipeline, such setting will lower the NGS sensitivity and miss clinically-relevant mutations.

What about the missing false-negative mutations?

Insufficient sequencing depth (coverage) and reference bias in NGS can miss true mutations and increase false-negative mutation rates. It is reported that the false-negative rate varies between 6% and 18%, much higher compared to the <3% false-positive rate, but much less attention is given to the false-negative mutations.

The sensitivity for detecting variant allele frequency (VAF) can vary from 5% to 1% (a 0.1% sensitivity has been also claimed). Some of these variants may have clinical relevance but are missed by NGS. Sanger sequencing is a great tool for confirming false-positive mutations but fails to confirm the missing false-negative mutations, although the new development claims that Sanger sequencing can reach 5% sensitivity. For confirmation of these NGS-identified false-negative mutations, other more sensitive methods need to be used.

XNA technology used to detect NGS false-negative mutations

The use of XNA technology suppresses normal DNA amplification but selectively enriches variant amplification in a PCR reaction. It is a powerful technology for identifying mutations in a small population of tumor cells in the background of a large normal cell population. We have successfully used the XNA technology in NGS to identify multiple cancer gene mutations missed by regular NGS in patient samples.

In addition to its use in NGS to confirm missed mutations by NGS, qPCR assays using XNA has also been used to identify important mutations in KRAS, EGFR, NRAS, and BRAF using qPCR. If the exact nature of the mutation needs to be known, the PCR product using the qPCR product as a template can be sent for Sanger sequencing.