A simple method to estimate the in-house limit of detection for genetic mutations with low allele frequencies in whole-exome sequencing analysis by next-generation sequencing

Takumi Miura,Yoji Sato,Satoshi Yasuda

doi:10.1186/s12863-020-00956-x

Abstract

BackgroundNext-generation sequencing (NGS) has profoundly changed the approach to genetic/genomic research. Particularly, the clinical utility of NGS in detecting mutations associated with disease risk has contributed to the development of effective therapeutic strategies. Recently, comprehensive analysis of somatic genetic mutations by NGS has also been used as a new approach for controlling the quality of cell substrates for manufacturing biopharmaceuticals. However, the quality evaluation of cell substrates by NGS largely depends on the limit of detection (LOD) for rare somatic mutations. The purpose of this study was to develop a simple method for evaluating the ability of whole-exome sequencing (WES) by NGS to detect mutations with low allele frequency. To estimate the LOD of WES for low-frequency somatic mutations, we repeatedly and independently performed WES of a reference genomic DNA using the same NGS platform and assay design. LOD was defined as the allele frequency with a relative standard deviation (RSD) value of 30% and was estimated by a moving average curve of the relation between RSD and allele frequency.ResultsAllele frequencies of 20 mutations in the reference material that had been pre-validated by droplet digital PCR (ddPCR) were obtained from 5, 15, 30, or 40 G base pair (Gbp) sequencing data per run. There was a significant association between the allele frequencies measured by WES and those pre-validated by ddPCR, whose p-value decreased as the sequencing data size increased. By this method, the LOD of allele frequency in WES with the sequencing data of 15 Gbp or more was estimated to be between 5 and 10%.ConclusionsFor properly interpreting the WES data of somatic genetic mutations, it is necessary to have a cutoff threshold of low allele frequencies. The in-house LOD estimated by the simple method shown in this study provides a rationale for setting the cutoff.

Highlights

Next-generation sequencing (NGS) has profoundly changed the approach to genetic/genomic research
Sequencing coverage of on-target regions To assess the performance of a measurement system for Allele frequency (AF) of genomic variants by NGS, we carried out a performance test for NGS using genomic DNA with 20 mutations, whose AFs had been determined by digital droplet PCR in advance, as a reference material for NGS
After performing whole-exome sequencing (WES) and mapping to the reference sequence, we investigated the relationship between the precision of the measured AFs and the amount of sequencing data

Summary

Introduction

Next-generation sequencing (NGS) has profoundly changed the approach to genetic/genomic research. The quality evaluation of cell substrates by NGS largely depends on the limit of detection (LOD) for rare somatic mutations. In the past few years, NGS techniques have been used for noninvasive prenatal testing to analyze fetal aneuploidy [6] They are utilized for various other applications, including the detection of mutations in tumor-related genes for quality control of cell therapy products or genetically modified cell products [7, 8]. Despite the increasing use of NGS for detecting low-level somatic mutations in clinical tissue samples and cell substrates for biopharmaceuticals, including cell therapy products and genetically modified cell products, the LOD of NGS for allele frequencies (AFs) is not verified often in each laboratory. Prior to detecting mutations with low AFs, researchers should have a cutoff threshold to eliminate false-positive results, using the determined or estimated LOD based on a clear understanding of the performance and limitations of their NGS-based methodology in their laboratories

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