Abstract Background The sequencing-by-synthesis technology by Illumina, Inc. enables efficient and scalable readouts of mutations from genomic data. To enhance sequencing speed and efficiency, Illumina has shifted from the four-color base calling chemistry of the HiSeq series to a two-color fluorescent dye chemistry in the NovaSeq series. Benchmarking sequencing artifacts due to biases in the newer chemistry is important to evaluate the quality of identified mutations. Results We re-analyzed a series of whole-genome sequencing experiments in which the same samples were sequenced on the NovaSeq 6000 (two-color) and HiSeq X10 (four-color) platforms by independent groups. In several samples, we observed a higher frequency of T-to-G and A-to-C substitutions (“T>G”) at the read level for NovaSeq 6000 versus HiSeq X10. As the per-base error rate is still low, the artifactual substitutions have a negligible effect in identifying germline or high variant allele frequency (VAF) somatic mutations. However, such errors can confound the detection of low-VAF somatic variants in high-depth sequencing samples, particularly in studies of mosaic mutations in normal tissues, where variants have low read support and are called without a matched normal. The artifactual T>G variant calls disproportionately occur at NT[TG] trinucleotides, and we leveraged this observation to bioinformatically reduce the T>G excess in somatic mutation callsets. Conclusions We identified a recurrent artifact specific to the Illumina two-color chemistry platform on the NovaSeq 6000 with the potential to contaminate low-VAF somatic mutation calls. Thus, an unexpected enrichment of T>G mutations in mosaicism studies warrants caution.