Abstract
In the past decade, treatments for tumors have made remarkable progress, such as the successful clinical application of targeted therapies. Nowadays, targeted therapies are based primarily on the detection of mutations, and next-generation sequencing (NGS) plays an important role in relevant clinical research. The mutation frequency is a major problem in tumor mutation detection and increasing sequencing depth is a widely used method to improve mutation calling performance. Therefore, it is necessary to evaluate the effect of different sequencing depth and mutation frequency as well as mutation calling tools. In this study, Strelka2 and Mutect2 tools were used in detecting the performance of 30 combinations of sequencing depth and mutation frequency. Results showed that the precision rate kept greater than 95% in most of the samples. Generally, for higher mutation frequency (≥20%), sequencing depth ≥200X is sufficient for calling 95% mutations; for lower mutation frequency (≤10%), we recommend improving experimental method rather than increasing sequencing depth. Besides, according to our results, although Strelka2 and Mutect2 performed similarly, the former performed slightly better than the latter one at higher mutation frequency (≥20%), while Mutect2 performed better when the mutation frequency was lower than 10%. Besides, Strelka2 was 17 to 22 times faster than Mutect2 on average. Our research will provide a useful and comprehensive guideline for clinical genomic researches on somatic mutation identification through systematic performance comparison among different sequencing depths and mutation frequency.
Highlights
In the past decade, treatments for tumors have made remarkable progress, such as the successful clinical application of targeted therapies
Sites with completely different homozygous genotypes between YanHuang No.1 (YH-1) and NA12878 were selected into true mutations set, the percentage of YH-1 can be taken as the somatic mutation frequency for the mixed sample
The depths of mixed bam files were grouped into 100X, 200X, 300X, 500X and 800X, and for each depth, 1%, 5%, 10%, 20%, 30% and 40% of YH-1 was mixed with NA12878, separately
Summary
Treatments for tumors have made remarkable progress, such as the successful clinical application of targeted therapies. Nowadays, targeted therapies are based primarily on the detection of mutations, and next-generation sequencing (NGS) plays an important role in relevant clinical research. The mutation frequency is a major problem in tumor mutation detection and increasing sequencing depth is a widely used method to improve mutation calling performance. Strelka[2] and Mutect[2] tools were used in detecting the performance of 30 combinations of sequencing depth and mutation frequency. Our research will provide a useful and comprehensive guideline for clinical genomic researches on somatic mutation identification through systematic performance comparison among different sequencing depths and mutation frequency. The result of detecting somatic mutations can www.nature.com/scientificreports be influenced by many factors, such as sequencing depth, the proportion of pathological mutated subclone and mutation calling software. We choose Mutect[2] and Strelka[2] for somatic mutation-calling pipeline in the present study
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