DNA mutations are the basis of all heritable biological variation. Some variation gives rise to beneficial traits, many are neutral or nearly neutral, and some result in deleterious phenotypes. While it is commonly believed that DNA mutations affect evolutionary phenotypic trajectories over millions of years, scientific advances have shown that this evolution can occur over much shorter time scales; years, days or even hours. The ability to pinpoint novel mutations in cell populations is of particular interest to many diverse areas of science, from cancer evolution and viral evolution to personalized or predictive medicine. Unfortunately, precise measurement of DNA mutation by high throughput sequencing is hindered by high sequencing error rates. Various strategies have been employed to get around this limitation, but most have drawbacks including high labor costs or low throughput.
Researchers at Arizona State University in conjunction with a collaborator at Mississippi State University have developed a novel protocol for sequencing genomic DNA and obtaining mutation rate estimates with a sequencing resolution floor of at least ~1*10-7 per base, or miscall per 10 million bases, potentially lower with more engineering. Circle-seq detects DNA mutation rates significantly lower than the mutation burdens expected in viruses, somatic cells or cancer cells over time. When tested, this protocol recovered some signal of the mutation spectrum of MMR- E. coli, which has a mutation rate of ~3*10-8.
This protocol requires less input DNA, is faster, and has reduced sequencing errors to provide an economic and fairly rapid means of estimating DNA mutation burden, and DNA mutation rate.
Potential Applications
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High Throughput nucleic acid sequencing and mutation rate estimation
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Personalized medicine
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Predictive medicine
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Cancer Diagnostics – tumor heterogeneity
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Viral evolution
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DNA Damage assays
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Antibiotic resistance
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Research
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Benefits and Advantages
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Reduces sequencing error rate 76x-1750x, depending on preferred benchmark.
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Only requires a single biopsy of tissue
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Increased reliability and reproducibility
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Leaner protocol that takes 1/20th the input DNA, half the execution time, produces micrograms of DNA for sequencing with reduced sequencing error rates.
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Reduces errors introduced to the library prep in vitro
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Streamlined and potentially automatable
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Requires less expensive reagents and has an acceptable resolution level for measuring many biological mutation rates
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Less laborious vs competing techniques
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For more information about the inventor(s) and their research, please see