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