Research Article: False-negative errors in next-generation sequencing contribute substantially to inconsistency of mutation databases

Date Published: September 12, 2019

Publisher: Public Library of Science

Author(s): Young-Ho Kim, Yura Song, Jong-Kwang Kim, Tae-Min Kim, Hye Won Sim, Hyung-Lae Kim, Hyonchol Jang, Young-Woo Kim, Kyeong-Man Hong, Amanda Ewart Toland.


More than 11,000 laboratories and companies developed their own next-generation sequencing (NGS) for screening and diagnosis of various diseases including cancer. Although inconsistencies of mutation calls as high as 43% in databases such as GDSC (Genomics of Drug Sensitivity in Cancer) and CCLE (Cancer Cell Line Encyclopedia) have been reported, not many studies on the reasons for the inconsistencies have been published. Methods: Targeted-NGS analysis of 151 genes in 35 cell lines common to GDSC and CCLE was performed, and the results were compared with those from GDSC and CCLE wherein whole-exome- or highly-multiplex NGS were employed.

In the comparison, GDSC and CCLE had a high rate (40–45%) of false-negative (FN) errors which would lead to high rate of inconsistent mutation calls, suggesting that highly-multiplex NGS may have high rate of FN errors. We also posited the possibility that targeted NGS, especially for the detection of low-level cancer cells in cancer tissues might suffer significant FN errors.

FN errors may be the most important errors in NGS testing for cancer; their evaluation in laboratory-developed NGS tests is needed.

Partial Text

Whereas classical genetic tests measure limited base changes or structural changes in DNA, recently introduced next-generation sequencing (NGS) technology can examine millions of DNA variants at a time. According to the American Clinical Laboratory Association (ACLA), over 11,000 laboratories and companies in the USA have developed their own NGS tests [1]. Despite widespread adoption of NGS technology for the purposes of clinical diagnosis, high error rates on various NGS platforms has been reported (0.26–12.86%) [2]. In a study with 20,000 samples, the NGS false-positive rate was 1.3%, the authors suggesting that Sanger confirmation is required for NGS panel testing [3]. Some of the errors seem to be related to the NGS instrumentation itself, the SOLiD platform having shown an erroneous variant calling rate as high as 20–40% in a study [4]. Currently, in the USA, the clinical laboratories for NGS testing are overseen by the Centers for Medicare and Medicaid Services (CMS) according to the Clinical Laboratory Improvement Amendments (CLIA) regulations [5]. The Food and Drug Administration (FDA) has finalized a guidance document to accelerate the establishment of a regulatory approach for NGS testing [6,7], though the actual regulations are still debated. To establish any firm regulatory rules, further studies on the reproducibility or reliability of NGS results are needed.

We performed T-NGS to investigate the reasons for inconsistencies in mutation calls between GDSC and CCLE, which databases had been constructed by whole-exome- or equivalently highly-multiplex NGS. In our sequencing-NGS analysis for 151 genes in 35 cell lines, the P-FN rates in GDSC or CCLE were as high as 42–51%, whereas the P-FP rates were only 5.0–8.0%, suggesting that FN errors may be the most important reason for the discrepancy between databases and for errors in highly-multiplex-NGS analyses.

FN errors may be the most important reason for the errors or inconsistencies in highly-multiplex-NGS tests for mutation detection in cancer. Also in T-NGS tests, FN errors may be one of the most important factors for the analysis of cancer tissues with low-percentage cancer cells designed for extremely high-sensitivity detection of mutations.